Patent Grant
8586726
All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 17, 2010, is named 19240US3.txt, and is 1,134,968 bytes in size.
A lengthy table (for example, Table 11) is referenced in this application and has been filed as an Appendix to this invention. The specification of the application contains reference to the single table, Table 11, which consists of more than 51 pages, and is hereby incorporated by reference in its entirety. Table 11 contains information encompassing gene sequences pertaining to the analysis of cross-species conservation for miRNAs. The Table displays results for conservation of full-length mature miRNA sequences, and seed of the mature sequence.
Various nucleic acid species are capable of modifying gene expression. These species include antisense RNA, siRNA, microRNA, RNA and DNA aptamers, anatgomirs, and decoy RNAs. Each of these nucleic acid species can inhibit target nucleic acid activity, including gene expression.
MicroRNAs (miRNAs, miR5) are 20-23 nucleotides (nt) RNA molecules that are produced by the processing of a larger enclosing stem-loop structure (>50 bp), called precursors, by cellular enzymes. miRNAs are processed from hairpin precursors of 70 nt (pre-miRNA) which are derived from primary transcripts (pri-miRNA) through sequential cleavage by the RNAse III enzymes drosha and dicer. miRNAs target the messenger RNA of other genes by binding to their 3′ UTR and interfering with their translation or causing degradation by enzyme targeting double-stranded RNA. miRNAs are non-coding RNAs (ncRNAs) that exist in a variety of organisms, including mammals, and are conserved in evolution. Many miRNAs tend to be clustered and transcribed as polycistrons and often have similar spatial temporal expression patterns. miRNAs have been implicated in various biological processes including developmental timing, differentiation, apoptosis, cell proliferation, organ development, and metabolism
The invention is based, at least in part, on the discovery of newly-identified microRNAs from normal and tumor-related Human B cells. Accordingly, in one aspect, the invention features an isolated nucleic acid, wherein the nucleic acid: (a) consists of from about 14 to about 31 nucleotides in length; (b) exhibits expression in a human tissue; (c) has a nucleotide sequence not present in an exon; and (d) consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1-130 and 1094, and a nucleotide sequence which is about 97%, about 98%, or about 99% identical to a nucleic acid sequence comprising any one of SEQ ID NOS: 1-130 and 1094. In one embodiment, the nucleic acid is single stranded. In another embodiment, the nucleic acid is double-stranded. In a further embodiment, the human tissue comprises a lymphocyte. In some embodiments, the human tissue is a B cell. In other embodiments, the B cell comprises a Naïve B cell, a centroblast, a memory B cell, or a Ramos Burkitt Lymphoma cell.
An aspect of the invention provides for an isolated nucleic acid, wherein the nucleic acid: (a) consists of from about 14 to about 31 nucleotides in length; (b) exhibits expression in a human tissue; (c) has a nucleotide sequence not present in an exon; and (d) consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 131-401, and a nucleotide sequence which is about 97%, about 98%, or about 99% identical to a nucleic acid sequence having a SEQ ID NO: 131-401. In one embodiment, the nucleic acid is single stranded. In another embodiment, the nucleic acid is double-stranded. In a further embodiment, the human tissue comprises a lymphocyte. In some embodiments, the human tissue is a B cell. In other embodiments, the B cell comprises a Naïve B cell, a centroblast, a memory B cell, or a Ramos Burkitt Lymphoma cell.
The invention provides for an isolated nucleic acid that is complementary to a nucleic acid described in the aspects herein. In one embodiment, the nucleic acid is single stranded. In another embodiment, the nucleic acid is double-stranded.
The invention provides for an isolated nucleic acid that is complementary to all but 1, 2, 3, 4, or 5 nucleotides of the nucleic acids described in the aspects herein. In one embodiment, the nucleic acid is complementary to at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 contiguous nucleotides of a nucleic acid described in the aspects herein consisting from about 14 to about 31 nucleotides in length. In one embodiment, the nucleic acid is single stranded. In another embodiment, the nucleic acid is double-stranded.
The invention provides for a composition comprising one or more nucleic acids of described in the aspects herein, in any combination or permutation thereof. In one embodiment, the composition further comprises one or more carriers, excipients, solvents, bases, or a combination thereof.
The invention provides for a composition comprising one or more nucleic acids, wherein the one or more nucleic acids consist essentially of a nucleotide sequence of any one of SEQ ID NOS: 1-401 and 1094. In one embodiment, the composition further comprises one or more carriers, excipients, solvents, bases, or a combination thereof.
The invention provides for a method for modulating the activity of a target nucleic acid in a cell, wherein the method comprises contacting a cell with a nucleic acid described in the aspects herein. In one embodiment, the target nucleic acid is a mRNA, a mature miRNA, or a precursor to a mature miRNA. In another embodiment, the cell is a hematopoetic cell. In a further embodiment, the cell is a B cell. In some embodiments, the cell is in vitro or in vivo.
This invention provides for the discovery of a large number of new micro RNAs that have been identified to exist in normal Human B cells and/or in tumor-related Human B cells, using an integrated bioinformatics method and pipeline described herein.
Micro RNAs (miRNAs) are naturally-occurring 19 to 25 nucleotide transcripts found in over one hundred distinct organisms (such as nematodes, fruit flies, and humans). miRNAs can be processed from 60- to 70-nucleotide foldback RNA precursor structures, which are transcribed from the miRNA gene. The miRNA precursor processing reaction requires Dicer RNase III and Argonaute family members (Sasaki et al., 2003 Genomics 82, 323-330). The miRNA precursor or processed miRNA products are easily detected, and an alteration in the levels of these molecules within a cell can indicate a perturbation in the chromosomal region containing the miRNA gene.
At least 222 separate miRNA genes have been identified in the human genome. For example, 2 miRNA genes (miR15a and miR16a) have been localized to a homozygously deleted region on chromosome 13 that is correlated with chronic lymphocytic leukemia (Calin et al. (2002), Proc. Natl. Acad. Sci. USA 99:15524-29). However, the distribution of miRNA genes throughout the genome, and the relationship of the miRNA genes to diverse chromosomal features, has not been systematically studied. A further review of miRNAs is provided in U.S. Pat. No. 7,232,806, U.S. Patent Application Publication No. 2006/0105360, and in the references: Landgraf et al., 2007, Cell 129: 1401-1414; Mendell, J T, 2005 Cell Cycle 4(9):1179-84; Shivdasani R A, 2006 Blood 108(12):3646-53; Hwang and Mendell, 2006 Br J Cancer 94(6):776-80; Hammond S M, 2006; Curr Opin Genet Dev. 16(1):4-9; Osada and Takahashi, 2007 Carcinogenesis 28(1):2-12; and Zhang et al., 2007 Dev Biol. 302(1):1-12, all of which are hereby incorporated by reference in their entirety.
All nucleic acid sequences herein are given in the 5′ to 3′ direction, for example the mature miRNA sequences listed in Table 1 (SEQ ID NOS: 1-401).
The unprocessed miRNA gene transcript is called a miRNA precursor (pre-miRNA) and comprises an RNA transcript of about 70 nucleotides in length. The pre-miRNA can be processed by digestion with an RNAse (such as, Dicer, Argonaut, or RNAse III, e.g., E. coli RNAse III)) into an active 19-25 nucleotide RNA molecule. This active 19-25 nucleotide RNA molecule is also called the processed miRNA gene transcript.
The active 19-25 nucleotide RNA molecule can be obtained from the miRNA precursor through natural processing routes (for example, using intact cells or cell lysates) or by synthetic processing routes (for example, using isolated processing enzymes, such as isolated Dicer, Argonaut, or RNAase III). The active 19-25 nucleotide RNA molecule can also be produced directly by biological or chemical syntheses, without having been processed from the miRNA precursor.
The invention provides for an isolated nucleic acid that: (a) consists of from about 14 to about 31 nucleotides in length; (b) exhibits expression in a human tissue; and (c) has a nucleotide sequence not present in an exon. In one embodiment, the isolated nucleic acid consists essentially of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1-401 and 1094, and a nucleotide sequence which is about 97%, about 98%, or about 99% identical to a nucleic acid sequence comprising any one of SEQ ID NOS: 1-401 and 1094. In some embodiments, the human tissue comprises a lymphocyte (for example, a human B cell). In other embodiments, the B cell comprises a Naïve B cell, a centroblast, or a memory B cell.
For example, an isolated nucleic acid, such as a miRNA of the invention, can be synthesized, or altered, or removed from the natural state through human intervention. A synthetic miRNA, or a miRNA partially or completely separated from the coexisting materials of its natural state, is considered isolated. An isolated miRNA can exist in substantially purified form, or can exist in a cell into which the miRNA has been delivered.
An isolated nucleic acid, such as a miRNA of the invention, can be obtained using a number of standard techniques utilized in the art. For example, the miRNA gene products can be chemically synthesized or recombinantly produced using methods known in the art. For example, a miRNA can be chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Rosetta Genomics (North Brunswick, N.J.), Pierce Chemical (part of Perbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), Ambion (Foster City, Calif., USA), and Cruachem (Glasgow, UK).
miRNA can also be expressed from recombinant circular or linear DNA plasmids using any suitable promoter. Suitable promoters for expressing RNA from a plasmid include, e.g., the U6 or H1 RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is within the skill in the art. Recombinant plasmids can comprise inducible or regulatable promoters for expression of the miRNA in cancer cells (such as hematopoietic cells, i.e., B cells). For example, a miRNA or a precursor miRNA of the invention (such as a miRNA molecule comprising any one of SEQ ID NOS: 1-401 and 1094) can be placed under the control of the CMV intermediate-early promoter, whereby the nucleic acid sequences encoding the miRNA molecule are located 3′ of the promoter, so that the promoter can initiate transcription of the miRNA gene product coding sequences.
miRNAs expressed from recombinant plasmids can be isolated from cultured cell expression systems by standard techniques. miRNAs which are expressed from recombinant plasmids can also be delivered to, and expressed directly in, the cancer cells. A miRNA can be expressed as an RNA precursor molecule from a single plasmid, and the precursor molecules are subsequently processed into functional miRNAs by a suitable processing system, including the processing systems naturally existing within a cell. Other suitable processing systems include, e.g., the in vitro Drosophila cell lysate system as described in U.S. Application Publication No. 2002/0086356 to Tuschl et al. and the E. coli RNAse III system described in U.S. Application Publication No. 2004/0014113 to Yang et al., which are herein incorporated by reference in their entireties.
Plasmids suitable for expressing a miRNA of the invention, methods for inserting nucleic acid sequences into the plasmid to express the miRNA of interest, and methods of delivering the recombinant plasmid to cells of interest are well-established and practiced in the art. See, for example, Zeng et al. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol, 20:446-448; Brummelkamp et al. (2002), Science 296:550-553; Miyagishi et al. (2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), Genes Dev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; and Paul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosures of which are herein incorporated by reference.
miRNA molecules of the invention can also be expressed from recombinant viral vectors. The RNA expressed from the recombinant viral vectors can either be isolated from cultured cell expression systems by standard techniques, or can be expressed directly in cancer cells (such as hematopoietic cells, i.e., B cells). For example, the recombinant viral vectors can comprise sequences that encode the miRNA molecule of interest and any suitable promoter for expressing the RNA sequences. Vectors can also comprise inducible or regulatable promoters for expression of the miRNA molecule in cells, such as cancer cell. As discussed previously, non-limiting examples of suitable promoters include the U6 or H1 RNA pol III promoter sequences, or the cytomegalovirus promoters. Selection of other suitable promoters is practiced by those of ordinary skill in the art.
Any viral vector that can harbor the nucleotide sequences for the miRNA molecules of the invention can be used. Non-limiting examples of such vectors include: vectors derived from adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus, and the like. The tropism of the viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. For example, AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes. An AAV vector expressing a serotype 2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsid gene to produce an AAV 2/5 vector. Techniques for constructing AAV vectors which express different capsid protein serotypes are within the skill in the art; see, e.g., Rabinowitz J. E. et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.
Recombinant viral vectors suitable for expressing miRNA molecules of the invention, methods for inserting nucleic acid sequences for expressing RNA in the vector, methods of delivering the viral vector to cells of interest, and recovery of the expressed RNA molecules are within the skill in the art. See, for example, Dornburg (1995), Gene Therap. 2:301-310; Eglitis (1988), Biotechniques 6:608-614; Miller (1990), Hum. Gene Therap. 1:5-14; and Anderson (1998), Nature 392:25-30, the entire disclosures of which are herein incorporated by reference. Useful viral vectors can be those derived from AV and AAV. A suitable AV vector for expressing a mRNA molecule of the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia et al. (2002), Nat. Biotech. 20:1006-1010, the entire disclosure of which is herein incorporated by reference. Suitable AAV vectors for expressing a miRNA molecule having a sequence shown in Table 1 (i.e., any one of SEQ ID NOS: 1-401), methods for constructing the recombinant AAV vector, and methods for delivering the vectors into target cells are described in Samulski et al. (1987), J. Virol. 61:3096-3101; Fisher et al. (1996), J. Virol., 70:520-532; Samulski et al. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.
Inhibition of RNA can effectively inhibit expression of a gene from which the RNA is transcribed. Inhibitors are selected from the group comprising: siRNA; interfering RNA or RNAi; dsRNA; RNA Polymerase III transcribed DNAs; ribozymes; and antisense nucleic acid, which can be RNA, DNA, or artificial nucleic acid. Also within the scope of the present invention are oligonucleotide sequences that include antisense oligonucleotides, antagomirs (also referred to as miRNA inhibitory nucleic acids), aptamers, and ribozymes that function to inhibit miRNA expression via purportedly binding to or degrading a miRNA molecule comprising any one of SEQ ID NOS: 1-401 and 1094.
The invention provides for a nucleic acid molecule that is substantially complementary to an isolated nucleic acid of the invention described above. “Substantially complementary” means that two sequences are substantially complementary that a duplex can be formed between them. The duplex can have one or more mismatches but the region of duplex formation is sufficient to down-regulate expression of the target nucleic acid. The region of substantial complementarity can be perfectly paired. In one embodiment, there can be nucleotide mismatches in the region of substantial complementarity. In one embodiment, the region of substantial complementarity will have no more than 1, 2, 3, 4, or 5 mismatches.
For example, an antagomir, an antisense RNA, a small interfering RNA (siRNA), a short hairpin RNA (snRNA), and the like) can be complementary to the guide strand of a miRNA having a nucleotide sequence shown in Table 1, positioned in the RNA silencing complex. This nucleic acid molecule can be single stranded or can be double stranded, and can inhibit the expression or activity of a miRNA molecule of the invention. In one embodiment, the nucleic acid molecule that inhibits a miRNA molecule of the invention (such as those described above) can complement at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 contiguous nucleotides of a miRNA having a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1-401 and 1094, and a nucleotide sequence which is about 97%, about 98%, or about 99% identical to a nucleic acid sequence comprising any one of SEQ ID NOS: 1-401 and 1094.
The invention also provides a method for modulating a target nucleic acid in a cell (for example, a miRNA molecule having a nucleotide sequence comprising any one of SEQ ID NOS: 1-401 and 1094) via contacting the cell with a nucleic acid of the invention (for example, those described above). For example, the nucleic acid can be substantially complementary to at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 contiguous nucleotides of a nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1-401 and 1094, and a nucleotide sequence which is about 97%, about 98%, or about 99% identical to a nucleic acid sequence comprising any one of SEQ ID NOS: 1-401 and 1094.
Expression of a miRNA molecule of the invention can be inhibited by an antisense oligonucleotide. Antisense oligonucleotides can comprise antisense DNA, RNA, and DNA/RNA molecule and act via altering the activity of the target RNA by binding to a target nucleic acid (such as a miRNA of interest) by means of RNA-RNA, RNA-DNA or RNA-PNA (protein nucleic acid) interactions (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat. No. 5,849,902). Antisense oligonucleotides suitable for use in the present methods are single-stranded nucleic acids (e.g., RNA, DNA, RNA-DNA chimeras, PNA) that generally comprise a nucleic acid sequence complementary to a contiguous nucleic acid sequence in a miRNA molecule. For example, the antisense oligonucleotide comprises a nucleic acid sequence that is 50-100% complementary, 75-100% complementary, or 95-100% complementary to a contiguous nucleic acid sequence in a miRNA molecule of the invention having a nucleic acid sequence of SEQ ID NO: 1-401, shown in Table 1. However, in some instances, an antisense molecule can form a loop and binds to a substrate nucleic acid which forms a loop. Thus, an antisense molecule can be complementary to two (or more) non-contiguous substrate sequences, or two (or more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence, or both. For a review of current antisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274, 21783-21789; Delihas et al., 1997, Nature, 15, 751-753; Stein et al., 1997, Antisense N A. Drug Dev., 7, 151; Crooke, 2000, Methods Enzymol., 313, 3-45; Crooke, 1998, Biotech. Genet. Eng. Rev., 15, 121-157; Crooke, 1997, Ad. Pharmacol., 40, 1-49.
Antisense DNA can also be used to target nucleic acid by means of DNA-RNA interactions, thereby activating RNase H, which digests the target nucleic acid in the duplex. The antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H to cleave a target nucleic acid. Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof. An RNase H activating region refers to a region (generally greater than or equal to 4-25 nucleotides in length, for example, from 5-11 nucleotides in length) of a nucleic acid compound capable of binding to a target nucleic acid to form a non-covalent complex that is recognized by cellular RNase H enzyme (see for example Arrow et al., U.S. Pat. No. 5,849,902; Arrow et al., U.S. Pat. No. 5,989,912). The RNase H enzyme binds to a nucleic acid compound-target nucleic acid complex and cleaves the target nucleic acid sequence.
Antisense nucleic acids can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above for the isolated miRNA molecules. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the miRNA molecules of the invention can be synthesized, e.g., by conventional phosphodiester techniques (Dallas et al., (2006) Med. Sci. Monit. 12(4):RA67-74; Kalota et al., (2006) Handb. Exp. Pharmacol. 173:173-96; Lutzelburger et al., (2006) Handb. Exp. Pharmacol. 173:243-59). Exemplary methods for producing and testing are within the skill in the art; see, e.g., Stein and Cheng (1993), Science 261:1004 and U.S. Pat. No. 5,849,902 to Woolf et al., the entire disclosures of which are herein incorporated by reference.
Antisense polynucleotides include, but are not limited to: morpholinos, 2′-O-methyl polynucleotides, DNA, RNA and the like. RNA polymerase III transcribed DNAs contain promoters, such as the U6 promoter. These DNAs can be transcribed to produce small hairpin RNAs in the cell that can function as siRNA or linear RNAs that can function as antisense RNA. The inhibitor can be polymerized in vitro, recombinant RNA, contain chimeric sequences, or derivatives of these groups. The inhibitor can contain ribonucleotides, deoxyribonucleotides, synthetic nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. In addition, these forms of nucleic acid can be single, double, triple, or quadruple stranded. (see for example Bass (2001) Nature, 411, 428 429; Elbashir et al., (2001) Nature, 411, 494 498; and PCT Publication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO 01/29058, WO 99/07409, WO 00/44914).
siRNA comprises a double stranded structure that can contain 15 to 50 base pairs, or 21 to 25 base pairs, and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. The siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions. The sense strand comprises a nucleic acid sequence which is substantially identical to a nucleic acid sequence contained within the target miRNA molecule. “Substantially identical” to a target sequence contained within the target mRNA refers to a nucleic acid sequence that is identical to the target sequence, or that differs from the target sequence by one or two nucleotides. The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded “hairpin” area.
The siRNA can also be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribonucleotides. One or both strands of the siRNA can also comprise a 3′ overhang. As used herein, a 3′ overhang refers to at least one unpaired nucleotide extending from the 3′-end of a duplexed RNA strand. For example, the siRNA can comprise at least one 3′ overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, or from 1 to about 5 nucleotides in length, or from 1 to about 4 nucleotides in length, or from about 2 to about 4 nucleotides in length. For example, each strand of the siRNA can comprise 3′ overhangs of dithymidylic acid (“TT”) or diuridylic acid (“uu”).
siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector, as described above for the miRNA molecules of the invention having a sequence shown in Table 1. Exemplary methods for producing and testing dsRNA or siRNA molecules are described in U.S. Patent Application Publication No. 2002/0173478 to Gewirtz, U.S. Patent Application Publication No. 2007/0072204 to Hannon et al., and in U.S. Patent Application Publication No. 2004/0018176 to Reich et al., the entire disclosures of which are herein incorporated by reference.
Expression of a miRNA molecule of the invention can also be inhibited by a short hairpin RNA (shRNA). The hairpin RNAs can be synthesized exogenously or can be formed by transcribing from RNA polymerase III promoters in vivo. Examples of making and using such hairpin RNAs for gene silencing in mammalian cells are described in, for example, Paddison et al., 2002, Genes Dev, 16:948-58; McCaffrey et al., 2002, Nature, 418:38-9; McManus et al., 2002, RNA, 8:842-50; Yu et al., 2002, Proc Natl Acad Sci USA, 99:6047-52). Such hairpin RNAs are engineered in cells or in an animal to ensure continuous and stable suppression of a desired gene. It is known in the art that siRNAs can be produced by processing a hairpin RNA in the cell.
Expression of a miRNA molecule of the invention can also be inhibited by a ribozyme. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA sequences (for example those shown in Table 1), followed by endonucleolytic cleavage. Engineered hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of a miRNA sequence shown in Table 1, are also within the scope of the present invention. Scanning the target molecule for ribozyme cleavage sites that include the following sequences, GUA, GUU, and GUC initially identifies specific ribozyme cleavage sites within any potential RNA target. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features such as secondary structure that can render the oligonucleotide sequence unsuitable.
The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides using, e.g., ribonuclease protection assays (see Romkes et al., 2005, Methods Mol. Biol.; 291:387-98; Dvorak et al., 2003, Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 147(2):131-5). The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, 1988, Nature, 334:585-591. Ribozymes can also include RNA endoribonucleases such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS or L-19 IVS RNA) and which has been described (see, e.g., Zaug, et al., 1984, Science, 224:574-578; Zaug and Cech, 1986, Science, 231:470-475; Zaug, et al., 1986, Nature, 324:429-433; published International patent application No. WO88/04300 by University Patents Inc.; Been and Cech, 1986, Cell, 47:207-216).
Both the antisense oligonucleotides and ribozymes of the present invention can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoamite chemical synthesis. Alternatively, antisense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters.
Alternatively, expression of a miRNA molecule of the invention can be inhibited by an antagomir. An antagomir is a single-stranded, double stranded, partially double stranded or hairpin structured chemically modified oligonucleotide agent that comprises at least 12 or more contiguous nucleotides substantially complementary to an endogenous miRNA or agents that include 12 or more contiguous nucleotides substantially complementary to a target sequence of an miRNA or pre-miRNA nucleotide sequence. The antagomir can be RNA, DNA, or a combination of RNA and DNA, an is antisense with respect to its target nucleotide sequence. An antagomir can target RNA, e.g., an endogenous pre-miRNA or miRNA of the subject. For example, the antagomir can target a miRNA having a nucleic acid sequence shown in Table 1. Exemplary methods for producing and testing antagomirs are discussed in U.S. Patent Application Publication No. 2007/0123482 and U.S. Patent Application Publication No. 005/0182005, in addition to Mattes et al., 2007 Am J Resp Cell Mol Biol 36: 8-12; Krützfeldt et al., 2007 Nuc Acid Res 35(9): 2885-2892, which are all incorporated by reference in their entireties.
Various modifications to the nucleic acid molecules of the present invention can be introduced as a means of increasing intracellular stability and half-life. Some modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5′ and/or 3′ ends of the molecule, or the use of phosphorothioate or 2′-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.
Expression of a miRNA molecule of the invention can be inhibited by an aptamer. Aptamer nucleic acid sequences are readily made that bind to a wide variety of target molecules. The aptamer nucleic acid sequences of the invention can be comprised entirely of RNA or partially of RNA, or entirely or partially of DNA and/or other nucleotide analogs. A nucleic acid aptamer is a nucleic acid or a nucleic acid-like molecule that is capable of binding to a specific molecule of interest with high affinity and specificity. A nucleic acid aptamer also can be a nucleic acid molecule that mimics the three dimensional structure of active portions of miRNAs. A nucleic acid-aptamer can be between about 9 and about 300 nucleotides or the like in length. More commonly, an aptamer is between about 30 and about 100 nucleotides or the like in length.
Aptamers are developed to bind specific ligands by employing known in vivo or in vitro selection techniques known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Nucleic acid-aptamers can be prepared by any known method, including synthetic, recombinant, and purification methods. Such methods are described in, for example, Ellington and Szostak (1990) Nature 346:818, Tuerk and Gold (1990) Science 249:505, James W., (2001) Current Opinion in Pharmacology, 1:540-546, Colas et al., (1996) Nature 380:548-550, U.S. Pat. No. 5,582,981; PCT Publication No. WO 00/20040; U.S. Pat. No. 5,270,163; Lorsch and Szostak (1994) Biochem. 33:973; Mannironi et al., (1997) Biochem. 36:9726; Blind (1999) Proc. Nat'l. Acad. Sci. USA 96:3606-3610; Huizenga and Szostak (1995) Biochem. 34:656-665; PCT Publication Nos. WO 99/54506, WO 99/27133, and WO 97/42317; and U.S. Pat. No. 5,756,291, all of which are incorporated by reference in their entireties.
Expression of a given miRNA molecule can be inhibited or decreased by inducing RNA interference of the miRNA molecule with an isolated double-stranded or single-stranded RNA molecule. For example, the miRNA inhibitor molecule can be those molecules discussed above, such as an antagomir, an antisense RNA, a small interfering RNA (siRNA), a short hairpin RNA (snRNA), and the like) which has at least about 75%, 80%, 90%, 95%, 98%, 99% or 100%, sequence homology to a portion of a miRNA molecule having a sequence shown in Table 1. For further discussion of modulation of miRNA expression, see: Lu et al., (2005) Adv Genet. 54:117-42; Leung and Whittaker (2005) Pharmacol Ther. 107(2):222-39; Takeshita and Ochiva (2006) Cancer Sci. 97(8):689-96; and Alexander et al., (2007) Arch Immunol Ther Exp (Warsz). 2007 May-June; 55(3):139-49.
A subject in need thereof, according to the invention, can refer to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals. In some embodiments, the subject can be a mouse, a rat, a bird, a dog, a cat, a cow, a horse, a sheep, or a pig. In exemplary embodiments, a mammal is a human.
Methods for determining RNA expression levels in cells from a biological sample are within the level of skill in the art. For example, tissue sample can be removed from a subject suspected of having cancer associated with a cancer-associated chromosomal feature by conventional biopsy techniques. In another example, a blood sample can be removed from the subject, and white blood cells isolated for DNA extraction by standard techniques. The blood or tissue sample should be obtained from the subject prior to initiation of radiotherapy, chemotherapy or other therapeutic treatment. A corresponding control tissue or blood sample can be obtained from unaffected tissues of the subject, from a normal human individual or population of normal individuals, or from cultured cells corresponding to the majority of cells in the subject's sample. The control tissue or blood sample is then processed along with the sample from the subject, so that the levels of miRNA molecules in cells from the subject's sample can be compared to the corresponding miRNA molecule levels from cells of the control sample. For example, the relative miRNA expression in the control and normal samples can be conveniently determined with respect to one or more RNA expression standards. The standards can comprise, for example, a zero miRNA expression level, the miRNA expression level in a standard cell line, or the average level of miRNA expression previously obtained for a population of normal human controls.
Suitable techniques for determining the level of RNA transcripts of a gene of interest in cells are within the skill in the art. According to one such method, total cellular RNA can be purified from cells by homogenization in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated, and DNA is removed by treatment with DNase and precipitation. The RNA molecules are then separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters by, e.g., the so-called “Northern” blotting technique. The RNA is then immobilized on the filters by heating. Detection and quantification of specific RNA is accomplished using appropriately labeled DNA or RNA probes complementary to the RNA in question. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7, the entire disclosure of which is incorporated by reference.
Suitable probes for Northern blot hybridization of a given miRNA molecule can be produced from the nucleic acid sequences provided in Table 1. Methods for preparation of labeled DNA and RNA probes, and the conditions for hybridization thereof to target nucleotide sequences, are described in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapters 10 and 11, the disclosures of which are herein incorporated by reference. For example, the nucleic acid probe can be labeled with, e.g., a radionuclide such as 3H, 32P, 33P, 14C, or 35S; a heavy metal; or a ligand capable of functioning as a specific binding pair member for a labeled ligand (e.g., biotin, avidin or an antibody), a fluorescent molecule, a chemiluminescent molecule, an enzyme or the like.
Probes can be labeled to high specific activity by either the nick translation method of Rigby et al. (1977), J. Mol. Biol. 113:237-251 or by the random priming method of Fienberg et al. (1983), Anal. Biochem. 132:6-13, the entire disclosures of which are herein incorporated by reference. Fienberg et al. provides a useful method for synthesizing 32P-labeled probes of high specific activity from single-stranded DNA or from RNA templates. For example, by replacing preexisting nucleotides with highly radioactive nucleotides according to the nick translation method, 32P-labeled nucleic acid probes can be prepared with a specific activity well in excess of 108 cpm/microgram. Autoradiographic detection of hybridization can then be performed by exposing hybridized filters to photographic film. Densitometric scanning of the photographic films exposed by the hybridized filters provides an accurate measurement of miRNA molecule levels. Using another approach, miRNA molecule levels can be quantified by computerized imaging systems, such the Molecular Dynamics 400-B 2D Phosphorimager available from Amersham Biosciences, Piscataway, N.J.
Where radionuclide labeling of DNA or RNA probes is not practical, the random-primer method can be used to incorporate an analogue, for example, the dTTP analogue 5-(N—(N-biotinyl-epsilon-aminocaproyl)-3-aminoallyl)deoxyuridine triphosphate, into the probe molecule. The biotinylated probe oligonucleotide can be detected by reaction with biotin-binding proteins, such as avidin, streptavidin, and antibodies (e.g., anti-biotin antibodies) coupled to fluorescent dyes or enzymes that produce color reactions.
In addition to Northern and other RNA blotting hybridization techniques, determining the levels of RNA transcripts can be accomplished using the technique of in situ hybridization. This technique requires fewer cells than the Northern blotting technique, and involves depositing whole cells onto a microscope cover slip and probing the nucleic acid content of the cell with a solution containing radioactive or otherwise labeled nucleic acid (e.g., cDNA or RNA) probes. This technique is well-suited for analyzing tissue biopsy samples from subjects. The practice of the in situ hybridization technique is described in more detail in U.S. Pat. No. 5,427,916, the entire disclosure of which is incorporated herein by reference. Suitable probes for in situ hybridization of a given miRNA molecule can be produced from the nucleic acid sequences provided in Table 1, as described above.
The relative number of miRNA transcripts in cells can also be determined by reverse transcription of miRNA transcripts, followed by amplification of the reverse-transcribed transcripts by polymerase chain reaction (RT-PCR). The levels of miRNA gene transcripts can be quantified in comparison with an internal standard, for example, the level of mRNA from a housekeeping gene present in the same sample, such as myosin or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). The methods for quantitative RT-PCR and variations thereof are within the skill in the art.
It is desirable to simultaneously determine the expression level of a plurality of different of miRNA molecules in a sample, for example determine the expression level of the transcripts of known miRNAs correlated with cancer or other cell division disorders (for example, a hematopoietic cell division disorder, such as a B cell lymphoma). Since examining cancer-specific expression levels for hundreds of miRNA molecules is time consuming, requires a large amount of total RNA (at least 20 μg for each Northern blot) and utilizes autoradiographic techniques that require radioactive isotopes, an oligolibrary in microchip format can be constructed containing a set of probe oligonucleotides specific for a set of miRNA molecules (for example, miRNA molecules having any one nucleic acid sequence of SEQ ID NOS: 1-401, shown in Table 1, or any one miRNA molecule of Table 7, Table 9, or Table 10).
A nucleic acid microchip array is a plurality of probe elements, each probe element comprising one or more nucleic acid molecules immobilized on one or more solid surfaces to which sample nucleic acids can be hybridized. Microarrays are known in the art and comprise a surface to which probes that correspond in sequence to gene products (e.g., cDNAs, mRNAs, cRNAs, polypeptides, and fragments thereof), can be specifically hybridized or bound at a known position. The microarray can be an array (i.e., a matrix) in which each position represents a discrete binding site for an RNA, and in which binding sites are present for products of most of the genes in the organism's genome, or a specific tissue or cellular subset of the organism. Here, the binding site can be a nucleic acid or nucleic acid analogue to which a cognate cDNA or RNA, such as miRNA molecule of the invention, can specifically hybridize. The nucleic acid or analogue of the binding site can be, e.g., a synthetic miRNA oligomer.
The nucleic acid or analogue is attached to a solid support, which can be made from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, or other materials. A useful method for attaching the nucleic acids to a surface is by printing on glass plates, as is described generally by Schena et al., 1995. See also DeRisi et al., 1996; Shalon et al., 1996; Schena et al., 1996. Each of these articles is incorporated by reference in its entirety.
The microchip is prepared from gene-specific oligonucleotide probes generated from known miRNAs. A nucleic acid array can contain two different oligonucleotide probes for each miRNA, one containing the active sequence and the other being specific for the precursor of the miRNA (for example, see Table 1). The array can also contain controls such as one or more mouse sequences differing from human orthologs by only a few bases, which can serve as controls for hybridization stringency conditions. tRNAs from both species can also be printed on the microchip, providing an internal, relatively stable positive control for specific hybridization. One or more appropriate controls for non-specific hybridization can also be included on the microchip. For this purpose, sequences are selected based upon the absence of any homology with any known miRNAs. For example, the array can also contain miRNA sequences found to be specific for human B cells. Non-limiting examples of such miRNA's include: mir-15, mir-17, mir-14, mir-124a-3, mir-99b, mir-167a, mir-167b, mir-129-1, mir-30c-2, mir-143, mir-27b, mir-125b-1, mir-128a, mir-140, mir-142, mir-191, mir-125b-2, mir-127, mir-129-2, mir-146a, mir-154, mir-185, mir-186, mir-322, mir-124a-1, mir-124a-2, mir-30c-1, mir-302a, and mir-99b.
The microchip can be fabricated by techniques known in the art. For example, probe oligonucleotides of an appropriate length, e.g., 40 nucleotides, are 5′-amine modified at position C6 and printed using commercially available microarray systems, e.g., the GeneMachine OmniGrid™ 100 Microarrayer and Amersham CodeLink™ activated slides. Labeled cDNA oligomer corresponding to the target RNAs is prepared by reverse transcribing the target RNA with labeled primer. Following first strand synthesis, the RNA/DNA hybrids are denatured to degrade the RNA templates. The labeled target cDNAs thus prepared are then hybridized to the microarray chip under hybridizing conditions, e.g. 6.times.SSPE/30% formamide at 25° C. for 18 hours, followed by washing in 0.75.times.TNT at 37° C. for 40 minutes. At positions on the array where the immobilized probe DNA recognizes a complementary target cDNA in the sample, hybridization occurs. The labeled target cDNA marks the exact position on the array where binding occurs, allowing automatic detection and quantification. The output consists of a list of hybridization events, indicating the relative abundance of specific cDNA sequences, and therefore the relative abundance of the corresponding complementary miRs, in the patient sample. According to one embodiment, the labeled cDNA oligomer is a biotin-labeled cDNA, prepared from a biotin-labeled primer. The microarray is then processed by direct detection of the biotin-containing transcripts using, e.g., Streptavidin-Alexa647 conjugate, and scanned utilizing conventional scanning methods. Images intensities of each spot on the array are proportional to the abundance of the corresponding miR in the patient sample.
Other methods for making microarrays (see U.S. Patent Application Publication No. 2006/0051771, which is incorporate by reference in its entirety), e.g., by masking (Fodor et al., 1991; Maskos and Southern, 1992), can also be used. In principal, any type of array, for example, dot blots on a nylon hybridization membrane (see Sambrook et al., 1989, which is incorporated in its entirety for all purposes), can be used, although, as will be recognized by those of skill in the art, very small arrays are useful because hybridization volumes will be smaller.
Labeled cDNA can be prepared from mRNA by oligo dT-primed or random-primed reverse transcription, both of which are well known in the art. Reverse transcription can be carried out in the presence of a dNTP conjugated to a detectable label, for example, a fluorescently labeled dNTP. Alternatively, isolated mRNA can be converted to labeled antisense RNA synthesized by in vitro transcription of double-stranded cDNA in the presence of labeled dNTPs (Lockhart et al., 1996, which is incorporated by reference in its entirety for all purposes). In alternative embodiments, the cDNA or aRNA probe can be synthesized in the absence of detectable label and can be labeled subsequently, e.g., by incorporating biotinylated dNTPs or rNTP, or some similar means (e.g., photo-cross-linking a psoralen derivative of biotin to RNAs), followed by addition of labeled streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the equivalent. Alternatively, cDNA or aRNA can be labeled indirectly by incorporation of 5-(3-aminoallyl) dNTPs or rNTPs to provide a amine reactive group for subsequent addition of label with any moiety bearing an N-Hydroxysuccinimide (NHS) ester.
Fluorescently labeled probes can be used, including suitable fluorophores such as fluorescein, lissamine, phycoerythrin, rhodamine (Perkin Elmer Cetus), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Fluor X (Amersham) and others (see, e.g., Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press San Diego, Calif.). It will be appreciated that pairs of fluorophores are chosen that have distinct emission spectra so that they can be easily distinguished. In another embodiment, a label other than a fluorescent label is used. For example, a radioactive label, or a pair of radioactive labels with distinct emission spectra, can be used (see Zhao et al., 1995; Pietu et al., 1996).
The analysis of microarray data can be accomplished using methods of statistical analysis known to those skilled in the art. For example, clustering analysis is commonly used for interpretation of microarray data. It provides both a visual representation of complex data and a method for measuring similarity between experiments. Some widely used methods for clustering microarray data include: hierarchical, K-means, and self-organizing map.
Southern blot hybridization techniques are also within the skill in the art. For example, genomic DNA isolated from a subject's sample can be digested with restriction endonucleases. This digestion generates restriction fragments of the genomic DNA that can be separated by electrophoresis, for example, on an agarose gel. The restriction fragments are then blotted onto a hybridization membrane (e.g., nitrocellulose or nylon), and hybridized with labeled probes specific for a given miRNA molecule(s). A deletion or mutation of these genes is indicated by an alteration of the restriction fragment patterns on the hybridization membrane, as compared to DNA from a control sample that has been treated identically to the DNA from the subject's sample. Probe labeling and hybridization conditions suitable for detecting alterations in gene structure or sequence can be readily determined by one of ordinary skill in the art. The miRNA nucleic acid probes for Southern blot hybridization can be designed based upon the nucleic acid sequences having SEQ ID NOS: 1-401, provided in Table 1, or any one miRNA molecule of Table 7, Table 9, or Table 10. Nucleic acid probe hybridization can then be detected by exposing hybridized filters to photographic film, or by employing computerized imaging systems, such the Molecular Dynamics 400-B 2D Phosphorimager available from Amersham Biosciences, Piscataway, N.J.
Human miRNAs are associated with different classes of chromosomal features that are subsequently associated with cancer (Xu and Li (2007) Chin Med J (Engl). 120(11):996-9; Bandres et al., (2007) DNA Cell Biol. 26(5):273-82). These cancers are purportedly partly caused by perturbing the chromosome or genomic DNA caused by the cancer-associated chromosomal feature, which can affect expression of oncogenes or tumor-suppressor genes located near the site of perturbation. A given cancer can be treated by restoring the level of miRNA expression associated with that cancer to normal. For example, if the level of miRNA expression is down-regulated in cancer cells of a subject, then the cancer can be treated by increasing the miRNA expression level. Alternatively, if the miRNA expression level is up-regulated in cancer cells of a subject, then the cancer can be treated by decreasing the miRNA expression level.
For example, the level of a miRNA in a cancerous or neoplastic cell of a subject (for example a hematopoietic malignancy or a hematopoietic neoplasm) is first determined relative to control cells. Techniques suitable for determining the relative level of a miRNA molecule in cells have been described above. If miRNA expression is down-regulated in the cancer or neoplastic cell relative to control cells, then the cancer or neoplastic cells are treated with an effective amount of a composition comprising a isolated miRNA molecule which is down-regulated (for example, a miRNA of the invention comprising any one of SEQ ID NOS: 1-401, as shown in Table 1, or any one miRNA molecule of Table 7, Table 9, or Table 10). If miRNA expression is up-regulated in cancer or neoplastic cells relative to control cells, then the cancer or neoplastic cells are treated with an effective amount of a composition that inhibits miRNA expression (for example, a miRNA of the invention comprising any one of SEQ ID NOS: 1-401, as shown in Table 1, or any one miRNA molecule of Table 7, Table 9, or Table 10).
One skilled in the art can also readily determine an appropriate dosage for the administration of an isolated miRNA molecule to a given subject. For example, a miRNA molecule can be administered to the subject once (e.g., as a single injection or deposition). Alternatively, a miRNA molecule of the invention can be administered once or twice daily to a subject for a period of from about two to about twenty-eight days, for example, from about seven to about ten days. Furthermore, the miRNA molecule of the invention can be co-administrated with another therapeutic, such as a chemotherapy drug. Where a dosage regimen comprises multiple administrations, the effective amount of the miRNA molecule administered to the subject can comprise the total amount of gene product administered over the entire dosage regimen.
The miRNA molecules of the invention comprising any one of SEQ ID NOS: 1-401, as shown in Table 1, or any one miRNA molecule of Table 7, Table 9, or Table 10, can be administered to a subject by any means suitable for delivering the miRNA molecules to cells of the subject, such as hematopoietic cells (either cancerous or neoplastic). For example, miRNA molecules can be administered by methods suitable to transfect cells (such as of the subject with the miRNA molecules of the invention. Transfection methods for eukaryotic cells (such as hematopoietic malignant cells or a hematopoietic neoplastic cells) are well known in the art, and include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate precipitation, and transfection mediated by viral vectors.
The compositions of this invention can be formulated and administered to inhibit a variety of disease states by any means that produces contact of the active ingredient with the agent's site of action in the body of a mammal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
Pharmaceutical compositions for use in accordance with the invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally can be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. (1985), the entire disclosure of which is herein incorporated by reference. For systemic administration, injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example, in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.
The present pharmaceutical formulations comprise the miRNA molecules of the invention comprising any one of SEQ ID NOS: 1-401, as shown in Table 1, or any one miRNA molecule of Table 7, Table 9, or Table 10 (e.g., 0.1 to 90% by weight), or a physiologically acceptable salt thereof, mixed with a pharmaceutically-acceptable carrier. The pharmaceutical formulations of the invention can also comprise the miRNA molecules of the invention comprising any one of SEQ ID NOS: 1-401, as shown in Table 1, or any one miRNA molecule of Table 7, Table 9, or Table 10, which are encapsulated by liposomes and a pharmaceutically-acceptable carrier. Useful pharmaceutically-acceptable carriers are water, buffered water, normal saline, 0.4% saline, 0.3% glycine, hyaluronic acid, and the like.
Pharmaceutical compositions of the invention can also comprise conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmolality adjusting agents, buffers, and pH adjusting agents. Suitable additives include physiologically biocompatible buffers (e.g., tromethamine hydrochloride), additions of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). Pharmaceutical compositions of the invention can be packaged for use in liquid form, or can be lyophilized.
For solid pharmaceutical compositions of the invention, conventional nontoxic solid pharmaceutically-acceptable carriers can be used; for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
For oral administration, the therapeutic compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration can be suitably formulated to give controlled release of the active agent. For buccal administration the therapeutic compositions can take the form of tablets or lozenges formulated in a conventional manner. For administration by inhalation, the compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflate or can be formulated containing a powder mix of the therapeutic agents and a suitable powder base such as lactose or starch.
The therapeutic compositions can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
Suitable enteral administration routes for the present methods include oral, rectal, or intranasal delivery. Suitable parenteral administration routes include intravascular administration (e.g. intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., peri-tumoral and intra-tumoral injection, intra-retinal injection, or subretinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application to the tissue of interest, for example by a catheter or other placement device (e.g., a retinal pellet or a suppository or an implant comprising a porous, non-porous, or gelatinous material); and inhalation. The miRNA molecules of the invention are administered by injection or infusion.
In addition to the formulations described previously, the therapeutic compositions can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the therapeutic compositions can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through nasal sprays or using suppositories. For topical administration, the compositions of the invention are formulated into ointments, salves, gels, or creams as generally known in the art. A wash solution can be used locally to treat an injury or inflammation to accelerate healing. For oral administration, the therapeutic compositions are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
A composition of the present invention can also be formulated as a sustained and/or timed release formulation. Such sustained and/or timed release formulations can be made by sustained release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 4,710,384; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,566, the disclosures of which are each incorporated herein by reference. The pharmaceutical compositions of the present invention can be used to provide slow or sustained release of one or more of the active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions. Suitable sustained release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the pharmaceutical compositions of the invention. Thus, single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelcaps, caplets, powders, and the like, that are adapted for sustained release are encompassed by the present invention.
In the present methods, the miRNA molecules of the current invention comprising any one of SEQ ID NOS: 1-401, as shown in Table 1, or any one miRNA molecule of Table 7, Table 9, or Table 10, can be administered to the subject either as naked RNA, in conjunction with a delivery reagent, or as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising sequences which expresses the gene product. Suitable delivery reagents for administration of the miRNA molecules include the Mims Transit TKO lipophilic reagent; lipofectin; lipofectamine; cellfectin; or polycations (e.g., polylysine), or liposomes.
The dosage administered will be a therapeutically effective amount of the composition sufficient to result in amelioration of symptoms of B cell lymphoma disease and can vary depending upon known factors such as the pharmacodynamic characteristics of the particular active ingredient and its mode and route of administration; age, sex, health and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment and the effect desired.
Toxicity and therapeutic efficacy of therapeutic compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (The Dose Lethal To 50% Of The Population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Therapeutic agents which exhibit large therapeutic indices are useful. Therapeutic compositions that exhibit some toxic side effects can be used.
Appropriate doses of small molecule agents depends upon a number of factors known to those or ordinary skill in the art, e.g., a physician. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention. Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram.
These methods described herein are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled artisan. Moreover, the effective amount of the compositions can be further approximated through analogy to compounds known to exert the desired effect.
The practice of aspects of the present invention can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). All patents, patent applications and references cited herein are incorporated in their entirety by reference.
A number of Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate the exemplary modes of making and practicing the present invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
Experimental Analysis:
We have used an established process for the identification of miRNAs candidates by sequencing size-fractionated cDNA libraries from three normal B cell populations, including Naïve B cells, Centroblasts and Memory B cells and from a Burkitt Lymphoma cell line (Ramos). Briefly, short RNAs (sRNA) extracted from the cells were linked to adaptor oligonucleotides, reverse transcribed, PCR amplified, cloned into an expression vector, and sequenced. Individual sequences, corresponding to short RNAs, were then analyzed bioinformatically to determine whether they would constitute appropriate miRNA candidates.
Bioinformatics Analysis:
The computational analysis starts with the identification of short RNA (sRNA) sequences from cloned cDNA sequences using the adaptor oligonucleotides as oriented markers. Then, each sRNA was matched to the human genome (ncbi36, October 2006) by using Megablast (NCBI).
A Bayesian evidence integration scheme was used to identify candidate sRNAs from partial matches to the human genome. Single and multiple base mismatches can occur for several reasons, including polymorphysms, sequencing errors, PCR errors that are more frequent in the 3′ and 5′ region of the miRNA cDNA, and RNA editing enzymes.
To identify sequences that correspond to real sRNAs, we used the following Bayesian approach to compute the posterior probability that a sequence is a candidate miRNA genomic match given specific base pair substitutions under the assumption that individual mutations are uncorrelated (Naïve Bayes). The individual p(mi|match) and p(mi) can be measured using matches to the miRNAs deposited in the miRNABase database. Sequences with p (match|m1, m2, . . . , mn)>0.5 were considered bona-fide matches to the human genome sequences and further processed together with the exact matching ones. m1, m2, . . . , mn:
Resulting sRNA genome locations, both for exact and partial matches, were analyzed and merged, if necessary, to remove overlapping sequences. For each sRNA hit a sequence from −80 bp upstream to +80 b.p. downstream of the sRNA match was selected leading to a set of candidate microRNA genes, including the full length precursors.
These longer sequences were then analyzed to determine if they can lead to the formation of precursor miRNA structures, including favorable energetics to for the established stem-loop secondary RNA structure required for further miRNA processing. Using a set of established mammalian miRNA's and data from the scientific literature the following criteria were established:
We do not apply highly popular conservation criteria because of:
Since the exact sites of transcription initiation and termination for miRNA genes is not known and since concentration and temperature can vary, it is important to consider not just the optimal folding variant but an entire distribution of variants near the optimal one. We used the Vienna RNA package to perform this type of analysis, with appropriate modifications to allow the analysis of suboptimal folding variants.
Candidate miRNA genes were filtered against non-coding RNA database and repeats database (repbase). Database of human non-coding RNA's is manually compiled and contains genes like ribosomal RNA's, snRNA, tRNA's, Y-RNA and others. Comparison performed using MEGABLAST program (NCBI).
Finally, candidate miRNAs emerging from this analysis were compared to miRNABase database to identify previously known miRNAs. Candidate miRNAs that were not identified in the miRNABase were considered. Diagrams illustrating these steps are included as
Validation:
Some of the newly identified miRNAs were tested by Northern in the corresponding population in which the sRNA was isolated.
Newly Identified miRNAs
The following have been identified as newly identified miRNAs (miRNAs) in one or more of the four B cell populations from which the size-fractionated cDNA libraries were isolated.
Preparation and Labeling of Decade Marker (Ambion #7778)
Decade marker is radio labeled using [γ-32P]ATP according to the manufacturer instructions.
Labeling of the RNA Carrier
20 pmol of #909 carrier RNA oligo is radio labeled using [γ-32P]ATP and T4 Polynucleotide kinase (NEB).
Purification of 18-26mers from Total RNA
A 15% denaturing polyacrylamide gel was prepared using the Sequagel System (National Diagnostics) following the manufacturer instructions. A metal plate was placed on the front of the gel and was pre-run at 50 W for 30 minutes using a running buffer of 0.5×TBE. Each RNA sample [long of total RNA] was subsequently spiked 1 pmol (2.5 n1) of [γ-32P] labeled-#909 carrier and an equal volume of 2×RNA loading buffer (Ambion) was added. For total RNA, extraction procedures like Trizol Reagent (Invitrogen) that purify all sizes RNA are recommended. Samples were then boiled for 5 minutes and were loaded with decade marker. After samples were loaded onto the gel, it was run for 3-4 hours at 50 W. After the gel was run, the apparatus was disassemble and one of the glass pieces was removed. The hot area of the gel was cut out and placed on the side of the lane for alignment with the markers. The gel was subsequently covered with a plastic wrap and was exposed to a phosphoimage screen for about 30 minutes to an 1 hour. The gel image was printed at 100% magnification and placed under the glass. It was then aligned with the hot spot that had been previously cut. The RNA band was cut approximately from 18mers up to 26mers using the marker and the 1 nt ladder below the carrier as reference. RNA was eluted from the gel using 2 ml of 0.3M NaCl in DEPC H2O, and the eluted sample was subsequently rotated overnight at 4° C. The supernatant was then recovered and 450 μl (max) was distributed into separate tubes. A 2× volume of 100% EtOH and 10 ng glycogen were added to each tube, which were then incubated at −20° C. for at least 2 hours. Samples were then spun for 30 minutes at 14,000 rpm, and were subsequently washed with 75% EtOH. The pellets were then air dried and dissolved in 10 μl, DEPC H2O.
3′-Adaptor Ligation and Purification
The following were first mixed in a tube and were then incubated at 25° C. for 6 hours:
15 μl, of 2× Loading Buffer (Ambion) was added to the samples and boiled for 5 minutes, which were then loaded onto a 12% denaturing polyacrylamide gel thata was prepared using the Sequagel System (National Diagnostics) following the manufacturer instructions. A metal plate was placed on the front of the gel and was pre-run at 50 W for 30 minutes using a running buffer of 0.5×TBE. The decade RNA marker and 1 μl, of [γ-32P] labeled-#909 carrier was then loaded into separated lanes and run for 3-4 hours at 50 W. After the gel was run, the apparatus was disassemble and the gel was exposed to a phosphoimage screen for 2-4 hours. The gel image was printed at 100% magnification and placed under the glass. The ligation product ranging approximately from 35 up to 42-45 nt was cut and RNA was eluted from the gel using 2 ml of 0.3M NaCl in DEPC H2O. The eluted sample was subsequently rotated overnight at 4° C. The supernatant was then recovered and 450 μl (max) was distributed into separate tubes. A 2× volume of 100% EtOH and 10 mg glycogen were added to each tube, which were then incubated at −20° C. for at least 2 hours. Samples were then spun for 30 minutes at 14,000 rpm, and were subsequently washed with 75% EtOH. The pellets were then air dried and dissolved in 12 μl DEPC H2O.
5′-Adaptor Ligation and Purification
The following were first mixed in a tube and were then incubated at 25° C. for 6 hours:
Samples can be stored at 4° C. after the 6 hour incubation until further processed. 10 μl of 2× Loading Buffer (Ambion) was added to the samples and boiled for 5 minutes, which were then loaded onto a 10% denaturing polyacrylamide gel thata was prepared using the Sequagel System (National Diagnostics) following the manufacturer instructions. A metal plate was placed on the front of the gel and was pre-run at 50 W for 30 minutes using a running buffer of 0.5×TBE. The decade RNA marker and 1 μl of [γ-32P] labeled-#909 carrier was then loaded into separated lanes and run for 3 hours at 50 W. After the gel was run, the apparatus was disassemble and the gel was exposed to a phosphoimage screen for 12-18 hours. The gel image was printed at 100% magnification and placed under the glass. The ligation product ranging approximately from 35 up to 48-62 nt was cut and RNA was eluted from the gel using 2 ml of 0.3M NaCl in DEPC H2O. The eluted sample was subsequently rotated overnight at 4° C. The supernatant was then recovered and 450 μl (max) was distributed into separate tubes. A 2× volume of 100% EtOH and 10 ng glycogen were added to each tube, which were then incubated at −20° C. for at least 2 hours. Samples were then spun for 30 minutes at 14,000 rpm, and were subsequently washed with 75% EtOH. The pellets were then air dried and dissolved in 20 μl DEPC H2O.
Reverse Transcription
The following were first mixed in a PCR tube and were then incubated at 80° C. for 2 minutes:
The sample was subsequently spun down to cool. Using the First Strand Synthesis System kit (Invitrogen), the following was added to the sample which was then incubated at 48° C. for 2 minutes:
3 μl of SuperScriptII (Invitrogen) was then added to the sample, which was further incubated at 48° C. for 1 hour. Samples can then be stored at −20° C. until further processing.
PCR Amplification
The PCR reaction was prepared as follows:
The sample was then amplified according to the following protocol:
The sample was then extracted using Phenol/CIA and was precipitated with sodium acetate. The sample was then spun for 30 minutes at 14,000 rpm, and was subsequently washed with 75% EtOH. The pellet was then air dried and dissolved in 45 DEPC H2O.
PACI Digestion
The digestion was prepared as follows and was subsequently incubate at 37° C. for 90 minutes:
The sample was then extracted using Phenol/CIA and was precipitated with sodium acetate. The sample was then spun for 30 minutes at 14,000 rpm, and was subsequently washed with 75% EtOH. The pellet was then air dried and dissolved in 15 μl DEPC H2O, Non-denaturing Loading Buffer was added to the sample and the sample was subsequently loaded onto a 12% non-denaturing acrylamide gel using pUC19/Sau3AI as a marker (Ambion #7760), which was run at 13 W for approximately 1 hr. The gel was then stained with 1:10,000 SybrGold (Molecular Probes #S-11494) in 0.5×TBE for 30 to 60 minutes. The smear of digested samples was then cut between 46 and 75 nt according to the marker size. Cut out samples were then eluted with 500 μl of 0.3M NaCl in 1.5 ml screw top tubes, rotating overnight at 4° C. Samples were spun briefly, and about 450 μl of eluted volume was recovered for each sample. The samples were then extracted using Phenol/CIA and were precipitated with sodium acetate. The samples were then spun for 30 minutes at 14,000 rpm, and were subsequently washed with 75% EtOH. The pellet was then air dried and dissolved in 10 μl DEPC H2O. If the amount of digested product on the gel appeared to be low, a second PCR amplification was required.
Second PCR Amplification
The PCR reaction was prepared as follows:
The sample was then amplified according to the following protocol:
The amount of product generated was examined via adding non-denaturing Loading Buffer to 5 μl of sample, which was then loaded onto a 12% non-denaturing acrylamide gel, and was subsequently run at 13 W for approximately 1 hr. Samples were cut out and eluted with 500 μl of 0.3M NaCl in 1.5 ml screw top tubes, rotating overnight at 4° C. Samples were spun briefly, and about 450 μl of eluted volume was recovered for each sample. The samples were then extracted using Phenol/CIA and were precipitated with sodium acetate. The samples were then spun for 30 minutes at 14,000 rpm, and were subsequently washed with 75% EtOH. The pellet was then air dried and dissolved in 45 DEPC H2O, Samples were further quantified, wherein amount of sampled needed to be within the range of 200-500 ng/μl.
BanI Digestion
The following digestion was prepared for each sample as follows, and were subsequently incubated at 37° C. for 2.5 hours:
Samples were then extracted using Phenol/CIA and were precipitated with sodium acetate. The samples were spun for 30 minutes at 14,000 rpm, and were subsequently washed with 75% EtOH. The pellet was then air dried and dissolved in 26 μl DEPC H2O.
Concatemerization
The following sample was mixed in a 0.5 or 0.2 ml tube as follows, and was incubated at 25° C. for 2 hours:
1 μl of dNTP as well as 1 μl of DNA Polymerase I was subsequently added, and the sample was then incubated at 16° C. for 1 hour. The sample was run on a 2% LM agarose-EtBr gel in 0.5×TBE with 100 bp Ladder loaded in the first lane. The concatamers were then cut starting from about 500 bp and above. Samples were then gel purified with QIAquick gel extraction kit (Qiagen cat. #28706), using the manufacturer's instructions.
Vector Digestion
The pCR2.1-TOPO (Invitrogen, 3.9 Kb) vector was digested according to the manufacturer's instructions for the EcoRV enzyme (NEB). The samples was then subjected to gel electrophoresis using a 1% agarose gel. The sample was then gel purified with QIAquick gel extraction kit (Qiagen cat. #28706), using the manufacturer's instructions.
Ligation
The ligation reaction was set up using the Rapid Ligation Kit (Roche cat. #11635379001) according to the procedure recommended by the manufacturer. 10 μl of the ligation product was used for transformation in high efficiency bacteria (One Shot OminMAX—Invitrogen cat. #C8540-03). X-gal/IPTG Ampicillin agarose plates were prepared fresh and a specified volume of the transformation reaction was spread onto the plates. Plated samples were then incubated overnight at 37° C. Colonies were then obtained the next day and grown in 30 μl of LB medium at 37° C. for 1 hour. Once DNA was obtained from the amplified bacteria, 2.5 μl was used as template for the colony PCR reaction.
Colony PCR
The PCR reaction was prepared as follows:
The sample was then amplified according to the following protocol:
1 μl of PCR product was examined on a 1.5% agarose gel and clones were selected carrying an insert longer than 350 bp. Samples were then confirmed by sequencing.
Oligonucleotides
5′ phosphate; 3′ none; includes PACI restriction site
5′ adenylated containing a pyrophosphate; 3′ modified terminal dideoxy-C(ddC) (available from IDT Inc. as the “miRNA cloning linker”); includes BanI restriction site.
It includes BanI restriction site.
It includes BanI restriction site.
It includes BanI restriction site.
It includes BanI restriction site.
Screening/Sequencing Primers:
The discovery of microRNAs (miRNAs) has added a new dimension to the mechanisms that regulate gene expression in normal cell development. Initial evidence also shows that structural or functional alterations of miRNAs are associated with tumorigenesis. The full set of microRNAs (miRNAs) in the human genome is not known. Since presently known miRNAs have been identified by virtue of their abundant expression in a few cell types, tissue-specific miRNAs may remain unrevealed. To understand the role of miRNAs in B-cell function and lymphomagenesis, short-RNA libraries were generated from normal human B cells at different stages of development (naïve, germinal-center, memory) and from a Burkitt lymphoma cell-line. The combination of cloning and computational analysis identified 401 miRNA (miRNome) expressed during normal B-cell development and/or in transformed B-cells. Most notably, this analysis identified 272 new miRNAs that were not previously reported. Numerous miRNAs are expressed in a stage-specific as well as transformation-specific fashion, suggesting specific functional roles. These results significantly increase the number of presently known miRNAs and provide a resource for the study of their role in B-cell development, immune function, and lymphomagenesis.
A new level of post-transcriptional regulation has been revealed with the discovery of microRNAs (miRNAs) a class of short-RNAs that impair translation or induce mRNA degradation by binding to the 3′ untranslated region of target mRNA1,2. The most recent release of the miRBase database (v.11.0)3′4 reports 839 human miRNAs, but the discovery of miRNAs is still an on-going process with variable predictions about the total number of miRNAs expressed in mammalian cells ranging from one thousand to several thousands5,6. The reported miRNAs have been identified from a limited number of cell types or from tissues whose cellular heterogeneity may favor the identification of ubiquitous and abundant miRNA. In fact, a recent report aiming to the identification of the miRNA expression profiles from 26 different mammalian tissues and cell types led to the discovery of only 12 new human miRNA7. These findings led to the conclusion that most miRNAs are known and that most of them are ubiquitously expressed. Nonetheless, additional analyses of purified cell populations may lead to the identification of tissue- and stage of differentiation-specific miRNAs, as has been the case for messenger RNAs.
The role of specific miRNAs in B-cell immunity and malignancy has only just begun to be elucidated. Using mouse models, miR-155 has been demonstrated to affect regulation of germinal center response through modulation of cytokine production8,9. Recently, miR-150 has been shown to target c-Myb, a critical transcription factor involved in the control of B cell differentiation10. In B cell lymphomas, 13q31 amplification has been associated with the over-expression of the miR-17-92 cluster and its enforced expression in a murine B cell lymphoma model showed a role in accelerating tumor development11. Furthermore, miR-15a and miR-16 have been implicated in the pathogenesis of B cell chronic lymphocytic leukemia (CLL)12,13.
To further explore the role of miRNAs in B cell function and lymphomagenesis, this study was aimed at identifying the miRNAs expressed (miRNome) in the human mature B-cell compartment, including naïve, germinal centers (GCs), and memory B cells. These B cell subpopulations are relevant for the development of antibody-mediated immunity as well as for tumorigenesis, since common human B-cell malignancies originate from the malignant transformation of GC B cells (most B-cell non-Hodgkin lymphomas, B-NHL), or naïve and memory B cells (mantle cell lymphoma and chronic lymphocytic leukemia)14,15. Using a combination of cloning and computational analysis, we report the identification of 401 miRNA representing the mature B cell miRNome, including 272 new miRNAs, and illustrate their pattern of expression during B cell differentiation and transformation.
Construction of Short-RNA Libraries from Human B Cell Sub Populations
Short-RNA libraries were generated by cloning RNA fractions of 18-30 nt from human centroblasts, naïve and memory B cells purified from tonsils as well as from the Burkitt lymphoma cell line Ramos, which is representative of malignant transformation of GC B cells (
The cloned sequences were matched to the human genome assembly from March 2006 (hg18) to retrieve the genomic regions from which the short-RNAs originated. One or more genomic locations were identified for approximately 80% of the cloned sequences considering both perfect matches and single mismatches. Consistent with previous observations, 3′-end mismatches were the most common and showed a clear preference for A in the last position (18. Approximately 546 short-RNA sequences did not align to the human genome according to the above criteria and are likely due to PCR errors introduced during the cloning procedure (
Computational Prediction of miRNA Precursors
In order to identify candidate miRNAs among the cloned sequences, we developed a computational pipeline aiming at the identification of potential miRNA precursors based on the investigation of their genomic location and folding characteristics (
The human miRNAs deposited in the miRBase database (v.11.0) were identified only at the end of the analysis and any lack of prediction was checked by matching the starting set with the final predictions. Overall, previously reported miRNA represented 32% of cloned and computationally validated mature miRNA. In addition, our analysis identified 146 previously reported precursors as well as 761 genomic locations containing precursors potentially coding for 272 new mature miRNA and 19 new precursors for 8 mature miRNAs deposited in the miRBase database (
Three hundred and thirty five sequences aligned to genomic regions which did not fulfill the criteria for miRNA precursors (
E. Coli
Each short-RNA is annotated according to the listed RNA species. Results shown in Table 2 refer only to short-RNAs with good-quality matches to the human genome. The same short-RNA may match to multiple databases and therefore the overall sum does not correspond to the total number of short-RNAs. The databases used in the analysis depicted in Table 2 is detailed in the Supplementary Methods section.
The remaining (236 sequences), however, mapped to genomic regions that lack annotations and may therefore represent a part of the transcriptome whose functions are unknown (Table 3A-3B and Table 4A-4B).
Interestingly, several of these non-annotated sequences (i.e. CU-5004, CU-5021, CU-6030, CU-6069) were cloned multiple times and showed differential expression across libraries, suggesting they may represent short-RNAs with characteristics distinct from those currently recognized in “classic” miRNAs.
In conclusion, the generation of short-RNA libraries from normal and neoplastic B cells led to the identification of 401 bona fide miRNAs as well as other short-RNA species of unknown function.
Abundance and Evolutionary Conservation
Previously reported miRNAs appeared to be more abundant than newly discovered miRNAs (
Regardless of their novelty, stage-specific miRNAs were observed with frequencies (defined as the fraction of the total pool of cloned miRNAs represented by a given miRNA) ranging between 0.03 and 0.6% of their respective libraries. The most abundant GC-associated miRNAs showed restricted expression in GC B cells and, if unaffected by transformation, in Ramos cells. However, in naïve and memory B cell libraries only the rarest miRNAs were truly exclusive in their expression and most of the non-GC-specific miRNAs were expressed in both naïve and memory cells albeit at different levels.
In order to investigate the presence of orthologous miRNA in other mammalian species, we relied on UCSC-provided Blastz pairwise alignments between human and target species and investigated conservation using two complementary methods, detailed in Supplementary Methods. The analysis was performed on the complete set of miRNAs deposited in the miRBase database and on the miRNAs (known and new) represented in the B cell libraries. Alignments of the human mature miRNA to its target species were required to have either perfect conservation of the entire mature miRNA sequence (
Validation of Newly Discovered miRNA
The newly identified miRNAs were investigated by Northern Blot analysis in order to validate their existence in vivo. Northern Blot analyses were performed using B cell lines and cells isolated from tonsil tissue obtained from multiple donors. Among 23 candidate miRNAs that have been cloned in any of the four libraries with 1-100 occurrences, 13 were detectable by Northern Blot (
Transcriptional and Post-Transcriptional Regulation
Most newly identified miRNAs showed a long abundant transcript (>150 nt) that might correspond to the primary miRNA transcript and a second transcript (˜60-80 nt) consistent with the precursor miRNA. As shown in
Distinct miRNA Signatures in Normal B Cells
miRNA representation in the four constructed libraries suggested differential expression of miRNAs during B cell differentiation and GC transit (
We also performed miRNA expression profiling of centroblasts, naïve and memory B cells (six donors/each) using a microarray representative of 723 known human miRNAs (miRBase v.10.1). Each B cell population showed a distinct miRNA expression profile. Consistent with the cloning data (
miRNA Signatures can Identify Subtypes of B Cell Malignancies
The miRNA library generated from Ramos BL cell line demonstrated that tumors can display specific miRNA expression signatures. To investigate whether these signatures can identify subtypes of B cell malignancies, miRNA expression profiling was performed using the same microarray platform on a panel of GC-derived malignancies including BL (8), DLBCL (16) and FL (10). Unsupervised clustering analysis of the tumor miRNA profiles was able to identify three major clusters enriched for samples belonging to each malignant phenotype (
Discussion
The combination of cloning procedures and computational tools led us to the identification of a large fraction of known as well as newly discovered miRNA expressed during B cell differentiation. These findings have general implications for the understanding of the total miRNA content of the human genome as well as for future studies on the role of miRNAs in B cell differentiation, function and lymphomagenesis.
The discovery of >250 new miRNAs and of their tissue-specific pattern of expression are in sharp contrast with previous reports that suggested, based on the discovery of only 12 new human miRNA7 from an analysis of 26 different organ systems and cell types, that most miRNAs are ubiquitously expressed and that most miRNAs have already been identified. These discordant results and conclusions may be partially due to the significantly higher number of clones per library sequenced in this study (3500 versus 1300 on average in7) which allowed the detection of low abundant miRNA species and to the criteria applied in the miRNA prediction (see Supplementary Methods).
The relatively lower degree of evolutionary conservation of tissue-specific miRNAs (
Two categories of short-RNAs were identified that could not be annotated as bona fide miRNAs. The first category is represented by those short-RNAs that could not be accurately mapped to the genome. Considering that a fraction of these RNAs were cloned multiple times and showed a stage-specific behavior, such short-RNAs do actually exist and that the lack of a match to the human genome may be due to polymorphisms, editing and other post-transcriptional modifications or to an incomplete/inaccurate sequencing of the corresponding genomic regions. The second category is represented by short-RNAs for which classic pre-miRNA structures could not be identified in the genome and no similarity to other non-coding RNA was found in the available databases. These short-RNAs may either be miRNA for which RNA secondary structure prediction algorithms failed to predict the correct hairpin structure or may represent new miRNA species of presently unknown mechanism of generation or other not yet described types of short-RNAs.
The stage specific expression of various miRNAs, especially in GC B cells, suggests highly specialized regulatory functions in B cell biology. The role of miRNAs that show cell type-specific functions in lymphocytes has just begun to be elucidated8-10,27. The miRNAs specifically associated to GC or non-GC B cells by either cloning or miRNA expression profiling (
Specificity in mature miRNA expression may be regulated at the transcriptional as well as at the post-transcriptional, i.e. pre-miRNA processing, level. Pre-miRNA accumulation in absence of a mature miRNA can occur in a cell type-restricted manner, suggesting the presence of a regulation mechanism at the pre-miRNA processing step. Both regulatory mechanisms may act during normal differentiation and may also be dysregulated during transformation as a consequence of genetic or epigenetic alterations22-24. The expanded B cell miRNome described here can be used to identify specific differences in miRNA expression in normal versus lymphoma cells that can guide searches for these tumor alterations.
miRNA expression profile differences between GC and non-GC B cells resembled those observed by expression profiling of coding genes28, consistent with the previous observation that miRNA profiling may be equally or more informative in discriminating cell phenotypes29. miRNA expression profiling, especially if including new B-cell specific miRNAs, may be useful in the differential diagnosis of lymphoid malignancies.
Materials and Methods
Generation of Short-RNA Libraries
Purification of naïve, memory and GC B cells was performed as previously reported28 using magnetic cell sorting of mononucleated cells obtained from human tonsils. Total RNA was purified using the Trizol Reagent (Invitrogen) following the manufacturer's indications. The short-RNA libraries were generated using an established protocol described in detail in30. Briefly, total RNA was separated on 15% polyacrylamide gel and the fragment corresponding to 18-28 nucleotides length was excised. The purified small RNAs were linked to adaptor oligonucleotides and gel purified. Upon adaptor ligation, RNA was reverse transcribed and cDNA was PCR amplified and cloned into pCR2.1-TOPO vector (Invitrogen). Sequencing was performed on colony PCR amplicons.
Computational Identification of Mature and Precursor MiRNAs
The bioinformatics miRNA analysis pipeline (
Orthology Analysis
The identification of putative orthologous sequences of known and predicted precursor and mature human miRNAs in chimp (panTro2), monkey (rheMac2), dog (canFam2) mouse (mm8) and rat (rn4) was performed using UCSC-provided Blastz31 pairwise alignments between human and target species. The details are reported in the Supplementary Methods.
miRNA Expression Profiling
The miRNA expression profiles were generated using the Human miRNA Microarray kit (Agilent Technologies) that allows detection of 723 known human (miRBase v.10.1) and 76 human viral miRNAs following the manufacturer's indications. Analysis of raw data was performed using the Feature Extraction Software 9.5.3.1 (Agilent Technologies). The dendrograms (
Northern Blot
Total RNA and small RNA fractions were purified using the Trizol Reagent (Invitrogen) and the PureLink miRNA Isolation Kit (Invitrogen), respectively, following the manufacturer's indications. Electrophoresis was performed on 15% denaturing polyacrylamide gel and then RNA was transferred on Duralon UV membrane (Stratagene) using a semidry transfer apparatus. Pre-hybridization and hybridization were performed in 5×SSC, 20 mM Na2HPO4 pH 7.2, 7% SDS, 3×Denhardt's Solution. Oligonucleotide probes were [γ32P]-ATP labeled by polynucleotide kinase (Fermentas). The list of oligonucleotides is reported in Table 5.
After over-night hybridization, membranes were washed at the same temperature in 3×SSC, 25 mM NaH2PO4 pH 7.5, 5% SDS, 10×Denhardt's Solution for 15-20′ and in 1×SSC, 1% SDS for 5′. Images were obtained by exposure to phosphoimager cassette and acquisition by Storm 840 Phosphoimager (Molecular Dynamics) and by film exposure for approximately 2 weeks.
Supplementary Methods
Bioinformatics Analysis of Short-RNA Libraries
The bioinformatics microRNA (miRNA) analysis pipeline includes (a) identification of short-RNAs from each library, (b) identification of exact and partial matches of the short-RNA sequences to the human genome, (c) testing each short-RNA genomic region for compatibility with hairpin secondary structures, (d) clustering genomic regions to predict mature miRNAs, (e) annotating and filtering short-RNAs and miRNAs candidates, (f) estimation of predicted miRNA frequencies in the libraries, (g) clustering short-RNAs that do not support miRNAs candidates.
a. Identification of Short-RNAs from Cloned cDNA Sequences:
Short-RNA sequences of length 15-30 bp were recovered from within cloned cDNA sequences using 8 bp adaptor oligonucleotides as oriented markers. An exact ≧6 bp match to the suffix of the 5′ daptor oligonucleotide and the prefix of the 3′ adaptor oligonucleotide were required. All short-RNAs of length 17 to 27 nt (length range of miRNAs deposited in the miRBase database v11.0) that are bounded by adaptors were reported, and short-RNAs containing adaptor fragments were tagged as lower confidence observations. miRNA candidates supported by low confidence short-RNAs were later evaluated individually and discarded by expert decision. The remaining ones are listed here: CU-1153 is supported by 29 short-RNAs including 1 low confidence; CU-1293 and CU-1079 are supported by low confidence short-RNAs including sequence fragments that could not originate from linkers.
b. Mapping to the Human Genome:
Each short-RNA was aligned to the human genome assembly from March 2006 (hg18) using WU-Blast. WU-Blast was ran locally optimal, with default word length 6, maximum allowed separation 2, no gaps, and no high scoring pair consistency. All WU-Blast reported matches were retrieved and mismatches marked. Only the genomic matches with the smallest number of mismatches were recorded, and we refer to them as short-RNA genomic locations.
c. Testing Short-RNA Genomic Locations for Hairpin Secondary Structures:
Short-RNA genomic locations were tested for compatibility with hairpin secondary structures. The criteria were established upon investigation of the characteristics of mammalian miRNA precursors deposited in the miRBase database (v.11.0)s1,s2. Only short-RNAs that have genomic locations compatible with a hairpin structure were used to define putative miRNAs and their precursors. A short-RNA genomic location was considered if the following criteria were satisfied: i) one or more hairpin structures were predicted in the genomic region starting at most 90 bases upstream and ending at most 90 bases downstream of the short-RNA genomic location; ii) the lowest free-energy predicted secondary structure for the containing region was a hairpin with maximum fold free energy of −8 joules, which is the maximum fold free energy observed for miRBase miRNAs; 110 the short-RNA genomic location could not overlap the predicted hairpin loop, which was required to be 3-20 bp long (the range of hairpin-loop lengths observed in the miRBase database); iv) the ratio between the number of complementary base pairs and the total number of base pairs in the stem was larger than 0.645, which is the minimum ratio observed in miRBase database. We used RNAfold 1.6, a part of the ViennaRNA package (http://www.tbi.univie.ac.at/˜ivo/RNA/), with temperature set to 37° C. to predict the lowest energy secondary RNA structure for each candidate genomic sequence.
d. Prediction of Mature miRNA:
Short-RNA genomic locations consistent with hairpin secondary structures (smirREGs) were clustered based on genomic region overlap, and smirREG clusters were pruned, split, merged, accepted, or eliminated iteratively. Clusters were first constructed from smirREGs corresponding to: (1) regions perfectly aligned to short-RNAs; (2) regions aligned to short-RNAs with 1-mismatch, represented by an ‘A’ in the last position; and (3) regions aligned to short-RNAs with 1-mismatch, where the associated short-RNAs had no perfect matches, were not used in (1) or (2) and were 1-mismatch away from short RNAs associated with smirREGs defined in (1) or (2). These smirREGs are major contributors to each region cluster. Regions associated with perfectly matching short-RNAs and 1-mismatch short-RNAs that were not used to define smirREG clusters, but overlapped 5 or fewer such clusters were tagged as minor contributors and added to the overlapping smirREG clusters. Each cluster was pruned or divided by identifying regions corresponding to short-RNAs where more than 25% of the region is supported by no more than 50% of the observations. For each cluster, all such regions were identified and first minor then major contributors with the largest ratio of unsupported portions to total length were iteratively removed until at least 75% of every contributing region was supported by more than 50% of the observations.
One exception is represented by CU-1088 cluster where a short-RNA was included despite the fact that only 70% of its sequence contributed to the majority observation. This exception was made because this short-RNA was the only one matching a known mature miRNA (miR-320a) and it was left out by the 75% rule.
Pruned smirREGs were merged and used to construct new and possibly overlapping clusters, but minor contributors were discarded. Finally, smirREG clusters were used to define putative mature miRNAs. The mature sequence was defined as the majority nucleotide in each position supported by more than 50% of the observations, with the genomic sequence allowed to break ties. SmirREG clusters were matched based on mature sequence containment. Clusters corresponding to a short-RNA set that is fully contained in a set corresponding to a matched cluster were eliminated, and matching clusters with partial containment were merged. The process was repeated until no elimination or merging was needed. Finally, a putative precursor was identified for each smirREG cluster following the procedure described in (c) with the added restriction that no more than 5 positions in the mature sequence were allowed to dangle off of the precursor region encapsulated by complimentary base pairs. When the mature region dangled off of the precursor region (but no more than 5 bases), the precursor was extended with non complimentary bases to include the mature region.
The mature miRNA prediction was followed by the elimination of incompatible predictions. Putative miRNAs whose predicted locations overlapped loops of known miRNAs or precursors of other higher-confidence predictions that could not form mature-star pairs were eliminated. Predicted miRNAs that were entirely composed of low confidence single-observation short-RNAs that contained linker fragments were also eliminated, with the exception of CU-1293 as described in (a). Mature miRNA predictions of length shorter than 17 nt or longer than 28 nt were discarded. Candidate miRNAs that were supported by a single observation were tagged as lower confidence predictions; some of these are likely to be miRNA, but others may be degradation products of previously unannotated RNA.
e. Annotation and Filtering of Candidate miRNA and sRNA:
Putative miRNAs and short RNAs were matched to several RNA databases (see below) via regular expression scans (for sequence databases) or genomic region containment (for databases that specify genomic regions). Databases identifying validated human mRNA, tRNA, snoRNA and yRNA were used to eliminate putative miRNA candidates with one observation and to annotate putative miRNAs with multiple observations. All putative miRNAs matching rRNAs were eliminated regardless of the number of observations because of the extreme abundance of rRNAs. Other RNA databases were used for annotation purposes only.
Precursor and mature miRNA as well as sRNA sequences were aligned to several sequence databases (see below) using the BLAST and MEGABLAST programs from NCBI. Candidate miRNA precursors showing a full match to non-coding RNAs (tRNA, rRNA, snoRNA, other nc-RNA) or to mRNA were disregarded. Predicted precursor and mature miRNAs were further classified as either “known” or “new” based on whether exact matches can be found in miRBAse database. Short-RNA sequences included in miRNA precursor, but not overlapping the mature miRNA, were considered as degradation products of miRNA precursor processing and marked as “miRNA other” (Table 6). All cloned short-RNA were annotated using the databases reported below and results are showed in Table 2.
E. coli
f. Estimation of Mature miRNA Frequencies:
Short-RNAs may contribute to more than one miRNA. In order to compare observation frequencies of predicted miRNAs we normalized the contribution of each supporting short-RNA to its associated predicted miRNA in each library. Short-RNAs supporting a single miRNA prediction were not affected, but the level of support of short-RNAs associated with several miRNAs was prorated. The normalization procedure was performed iteratively. First, for each library, we assigned an observation frequency to each miRNA, taken to be the sum of the library-specific observations across all of its supporting short-RNAs. Second, for each short-RNA observed in this library, we computed sum, the sum of the observation frequencies of the predicted miRNAs it supports. Then, short-RNA support for each miRNA was adjusted to be the number of its observations multiplied by the ratio between the observation frequency of this miRNA and sum. Finally, the frequency of each predicted miRNA was recalculated to be the sum of the adjusted frequencies of the short-RNAs supporting it. To compare the abundance of predicted miRNAs across libraries we normalized the frequencies of observations in each library to sum to 100%, comparing frequencies of observations in each library rather than raw observations. This normalization step was necessary due to the variability between the total number of observations across libraries.
g. Clustering Short-RNAs.
Short-RNAs that did not support predicted miRNAs were categorized according to the quality of their best alignments to hg18. These short-RNA were clustered following the procedure described in (c) but with no secondary structure requirements (Table 2 and Tables 9-10). Table 3 and Tables 9-10 were constructed from perfect matches and single mismatches, respectively, as described in (c). Tables 4 and 5 smirREGs were constructed from single-mismatches, double mismatches and three or more mismatches, respectively.
Estimation of Libraries Complexity
A bootstrap technique was used to estimate the total number of miRNAs expressed in each library and the number of short-RNAs must be sequenced to achieve a complete coverage. Bootstrapping is a statistical technique for estimating properties of an “estimator” by measuring those properties in multiple subsets of the sampless3,s4. Specifically, we estimated the distribution of mature miRNAs obtained by random sub-sampling different size short-RNA libraries from each complete library. For each size N=10, 20, . . . Nt, where Nt is the total number of short-RNAs in the library, we randomly sampled 1000 libraries of size N and computed the number r(N) of inferred miRNAs, resulting in a distribution p(r(N)) for which we could compute standard statistical parameters such as average, variance, mode and median. Based on this sampling, we can extrapolate p(r(N)) for increasing values of N to determine at which point it is no longer efficient to use larger values of N to increase miRNA coverage. To achieve this, we fitted the data to the parametric function ƒ(x)=K*(1−e−mx). Since we include both experimentally confirmed and putative mature miRNAs and since bootstrapping can produce optimistic results we expect that the estimated values constitute an upper boundary on the real library complexity.
Based on this analysis, we estimated that the total numbers of mature miRNAs are: 188 (naïve), 211 (memory), 219 (centroblasts) and 225 (Ramos). Thus, the libraries sequenced in this study cover respectively 84.0% (naïve), 87.2% (memory), 86.8% (centroblasts), and 85.8% (Ramos) of the expressed miRNAs in these cellular phenotypes.
Orthology and Conservation Analysis
We investigated conservation of known and predicted precursor and mature human miRNA in chimp (panTro2), monkey (rheMac2), dog (canFam2) mouse (mm8) and rat (rn4). We obtained 678 miRNA precursor sequences from miRBase (v.11.0); 677 (672 unique) mature miRNAs; and 170 (167 unique) star sequences. In total, we obtained 947 locations for mature and star mirBase sequences. We predicted 926 precursors of which 146 match miRBase precursors and 780 are newly predicted. Categorizing these by their corresponding mature sequences, 762 precursors correspond to mature miRNAs that are not included in the miRBase and 164 precursors are associated with 129 predicted miRNAs that match miRBase miRNAs. Of the 762 newly predicted precursors, one overlaps with a miRBase precursor and its corresponding predicted miRNA is a candidate star sequence; 19 precursors associated with 8 mature sequences listed in miRBase database.
Here, we predicted 762 miRNA genomic locations associated with unique mature miRNA sequences not included in miRBase. Of these 762 predicted miRNA genomic locations, one overlaps with a miRBase precursor and is a candidate star sequence. We identified 164 precursors associated with 129 predicted miRNAs matching the sequence of known miRNAs; 19 of the 164 precursors, associated with 9 known miRNAs, do not match known precursors. miRNA conservation has been repeatedly used to help identify putative miRNA mappings to genomes. To identify putative ortholog miRNAs we relied on UCSC-provided Blastz pairwise alignments between human and target speciess5. We used two related but complementary methods: (1) map the mature human miRNA to its ortholog location as specified by pairwise alignment; and (2) map the precursor of the human miRNA to its ortholog location as specified by pairwise alignment, expanding the human region to include at least 80 bases from both sides of the mature region, and identifying regions in the target that match the sequence of the mature human miRNA.
Method 1 is the simplest but fails to account for alignment inaccuracies and local mutations that may shift the position of the mature sequence in the target species. Method 2 accounts for locally imperfect Blastz mapping, but relies on conservation of larger regions that may not be subject to the same selective pressure as the mature miRNA. Alignment-based mapping of the human mature miRNA to its target were required to have either perfect conservation of the entire mature miRNA sequence or conservation of seeds composed of seven bases starting from the second position of the human mature sequence followed by conservation of 3 bases starting from the 12th, 13th, or 14th position as suggested bys6. We scanned the entire mapped ortholog region for a match to the human mature sequence or to its seed.
Comparison with Previously Reported MiRNA Prediction from Short-RNA Libraries
Landgraf et al.s7 used more restrictive miRNA characterizations for mature miRNA prediction, annotation and conservation. They required that at least 60% of the observations associated with a predicted miRNA align at the 5′ end. We made no such restriction, and some of the miRNAs with the highest number of observations in our libraries, such as CU-1026 and CU-1018, are supported by a high proportion of 5′-misaligned cloned sequences. Landgraf et al. eliminated predictions that can be derived from repeat sequences by excluding precursors that contain more than 30% repetitive elements and that match hg18 more than 10 times with at least 75% identity. We annotated mature sequences that match known repeats but did not eliminate them. Finally, Landgraf et al. formulated extensive criteria for sequence orthology, requiring sequence conservation greater than 75% in multiple alignments across vertebrates and either restricting the 20 5′-most misaligned nucleotides to be transitions or requiring 100% conservation in positions 2 through 8 and 90% conservation overall. We, as described above, simply considered full conservation and seed-based conservation, mapping human to primates, dog and rodents.
miRNome of Human Mature B Cells
Summary:
The full set of microRNAs (miRNAs) in the human genome is not known. Because presently known miRNAs have been identified by virtue of their abundant expression in a few cell types, many tissue-specific miRNAs remain unrevealed. To understand the role of miRNAs in B cell function and lymphomagenesis, we generated short-RNA libraries from normal human B cells at different stages of development (naïve, germinal center, memory) and from a Burkitt lymphoma cell line. A combination of cloning and computational analysis identified 178 miRNAs (miRNome) expressed in normal and/or transformed B cell libraries. Most notably, the B cell miRNome included 75 miRNAs which to our knowledge have not been previously reported and of which 66 have been validated by RNA blot and/or RT-PCR analyses. Numerous miRNAs were expressed in a stage- or transformation-specific fashion in B cells, suggesting specific functional or pathologic roles. These results provide a resource for studying the role of miRNAs in B cell development, immune function, and lymphomagenesis.
A new mechanism of post-transcriptional regulation has been revealed with the discovery of microRNAs (miRNAs), a class of short RNAs that impair translation or induce mRNA degradation by binding to the 3′ untranslated region of target mRNA ([Bartel, 2004] and [Kim, 2005]). A recent release of the miRBase database (v.11.0) ([Griffiths-Jones, 2006] and [Griffiths-Jones et al., 2006]) reports 847 human miRNAs. However, the discovery of miRNAs is still an on-going process with variable predictions about the total number of miRNAs expressed in mammalian cells ranging from one thousand to several thousands ([Bentwich et al., 2005] and [Miranda et al., 2006]). The reported miRNAs have been identified from a limited number of cell types or from tissues whose cellular heterogeneity may favor the identification of ubiquitous and abundant miRNA. In fact, a recent report aiming for the identification of miRNA expression profiles from a large panel of different mammalian tissues and cell types led to the discovery of only 12 previously unreported human miRNA (Landgraf et al., 2007). These findings led to the conclusion that most miRNAs are known and that most of them are ubiquitously expressed (Landgraf et al., 2007). Nonetheless, additional analyses of purified cell populations have led to the identification of tissue- and stage of differentiation-specific miRNAs in a few tissues, suggesting the existence of tissue-specific miRNA expression ([Calabrese et al., 2007] and [Cummins et al., 2006]).
The role of miRNAs in B lymphocyte development and B cell lymphomagenesis is largely unknown. A critical stage of the differentiation process leading to effector B cells is represented by the germinal centers (GC), the structures that develop when mature naive B cells encounter the antigen in the secondary lymphoid organs and are stimulated to proliferate and differentiate into GC centroblasts (CB). During the GC reaction, B cells undergo somatic hypermutation of their immunoglobulin-variable regions and class switch recombination. B cells that have acquired the ability to express high-affinity immunoglobulins are then positively selected and further differentiate into the final effectors of the humoral immune response, i.e., memory B cells and plasma cells (Klein and Dalla-Favera, 2008). Naive, GC, and memory B cells are also relevant targets of disease because each of these B cell subpopulations can be affected by malignant transformation leading to different types of lymphomas and leukemias ([Klein and Dalla-Favera, 2008] and [Kuppers and Dalla-Favera, 2001]).
Several initial observations suggest an important role of specific miRNAs in B cell function and malignancy. Via mouse models, miR-155 has been demonstrated to affect regulation of the GC response through modulation of cytokine production ([Rodriguez et al., 2007] and [That et al., 2007]) and by direct post-transcriptional regulation of the activation-induced cytidine deaminase (AID) ([Dorsett et al., 2008] and [Teng et al., 2008]). Recently, miR-150 has been shown to target MYB, a critical transcription factor involved in the control of B cell differentiation (Xiao et al., 2007). In B cell lymphomas, 13q31 amplification has been associated with the overexpression of the miR-17-92 cluster and its enforced expression in a murine B cell lymphoma model showed a role in accelerating tumor development (He et al., 2005). Furthermore, miR-15a and miR-16 have been implicated in the pathogenesis of B cell chronic lymphocytic leukemia (CLL) ([Calin et al., 2002] and [Calin et al., 2005]).
As a basis for a comprehensive analysis of the role of miRNAs in B cell function and lymphomagenesis, this study was aimed at identifying the miRNAs expressed (miRNome) in the human mature B cell compartment, including naive, GC, and memory B cells. By using a combination of cloning and computational analysis, we report the identification of 178 miRNAs representing the mature B cell miRNome, including 75 previously unreported miRNAs. The results show that normal B cell subpopulations are characterized by specific miRNA “signatures,” suggesting functional roles of miRNAs in B cell differentiation and transformation.
Results
Construction of Short-RNA Libraries from Human B Cell Subpopulations.
Short-RNA libraries were generated by cloning RNA fractions of 15-30 nt from human centroblasts, naive, and memory B cells purified from tonsils, as well as from the Burkitt lymphoma cell line Ramos, which is representative of malignant transformation of GC B cells. Approximately 3,500 sequences were analyzed from each library, corresponding to 13,788 total short-RNAs (2,632 nonredundant sequences). By using a bootstrap approach ([Harrell, 2001] and [Davison and Hinkley, 1997]), we estimated that more than 85% of the complexity of the libraries has been examined (
Mapping of Short-RNA Sequences to the Human Genome.
The cloned sequences were subjected to a computational analysis (see Supplemental Experimental Procedures described in Example 3) summarized in the flowchart illustrated in
Computational Prediction of Precursor and Mature miRNAs.
In order to identify candidate miRNAs among the cloned short-RNA sequences, we developed a computational pipeline aiming at the identification of potential miRNA precursors based on the investigation of their genomic location and folding characteristics (
Overall, the analysis identified 178 mature miRNAs, of which 103 were known and 75 were not previously reported, to our knowledge (Table 7 and
A sizeable number of sequences (334) aligned to genomic regions that did not fulfill the criteria for miRNA precursors (
In conclusion, the generation of short-RNA libraries from normal and neoplastic B cells led to the identification of 178 mature miRNAs cloned multiple times as well as other short-RNA species of unknown function.
E. Coli
Abundance and Evolutionary Conservation.
Previously reported miRNAs appeared to be generally more abundant than newly discovered miRNAs. Approximately 50% of previously reported miRNAs appeared in the libraries with more than 10 occurrences compared to 29% of the newly discovered miRNAs (
In order to investigate the presence of orthologous miRNA in other mammalian species, we relied on UCSC-provided Blastz pairwise alignments between human and target species and investigated conservation using two complementary methods, detailed in Supplemental Experimental Procedures. The analysis was performed on the complete set of miRNAs deposited in the miRBase database and on the miRNAs (known and new) represented in the B-cell libraries. Alignments of the human mature miRNA to its target species were required to have either perfect conservation of the entire mature miRNA sequence or conservation of seeds composed of seven bases starting from the second position of the human mature sequence followed by conservation of 3 bases starting from the 12th, 13th or 14th position as suggested by (Grimson et al., 2007) (
The majority of miRBase-miRNAs showed conservation across mammalian genomes, from primates to rodents. Conservation frequency mimicked known phylogenetic distances to human, with the highest conservation in chimp and lowest in rat. The conservation frequencies of known and newly identified miRNAs in B cells were similar in chimp (Pan troglodytes) and monkey (Macacus rhesus), especially when conservation requirements were restricted to the seed region of miRNAs. However, conservation frequencies in dog, mouse and rat were significantly divergent, with known miRNAs more likely to exhibit conservation than new candidate miRNAs (
Validation of Previously Unreported miRNAs.
All 75 newly identified miRNAs were investigated by RT-PCR analysis in order to independently validate their existence in vivo in B-cell lines and cells isolated from tonsils. Positive results were obtained in 66 of the cases (see
In order to gain preliminary evidence of the functionality of the previously unreported miRNAs, a small subset of these miRNAs which were fully validated at the expression level was tested for incorporation in the functional miRNA-mRNA complex by co-immunoprecipitation with Ago2 proteins (Mourelatos et al., 2002). The results showed that the RNA fraction associated with the Ago complex was indeed enriched for each of the four tested previously unreported miRNAs (
Indirect clues on the functionality of miRNAs may also be obtained analyzing the effect of stage-specific miRNAs on the corresponding transcriptome since most miRNAs have been showed to affect the expression of their targets albeit to a modest degree (Filipowicz et al., 2008). Toward this end, the targets of 15 previously unreported GC-over-expressed miRNAs were predicted by two algorithms (miRanda and RNA22) (John et al., 2004; Miranda et al., 2006) and were tested for enrichment in genes down-regulated in GC versus naïve B cells. Eleven out of 15 miRNA showed an increase (and only two a decrease) in their candidate target enrichment p-value for GC down-regulated genes compared to control populations (
In summary, previously unreported miRNAs identified by cloning and computational analysis were validated at the expression level by multiple detection methods. For a small subset tested, their incorporation in the Ago complex and their activity on the GC transcriptome suggests biological functionality.
Transcriptional and Post-Transcriptional Regulation.
Most newly identified miRNAs tested by RNA blot showed a long abundant transcript (>150 nt) likely corresponding to the primary miRNA transcript and a second transcript (˜60-80 nt) consistent with the precursor miRNA. As shown in
Taken together, these observations suggest that the expression of mature miRNAs may be affected by both transcriptional and post-transcriptional regulatory mechanisms.
Distinct miRNA Signatures in Normal B-Cell Subpopulations.
In order to further investigate whether specific miRNA regulation occurred in normal B-cell development or in transformed cells, miRNA representation was examined in libraries constructed from naïve, GC and memory B cells, as well as from the Ramos BL cell line. Differential expression of numerous known and newly identified miRNAs was evident during B-cell differentiation and GC transit as shown by hierarchical clustering using miRNA frequencies (defined as the fraction of the total pool of cloned miRNAs represented by a given miRNA in a library) obtained from the cloning data (
To independently validate results of the cloning experiment, miRNA expression profiling was performed of centroblasts, naïve and memory B cells (six donors/each) using a commercial microarray representative of 723 known human miRNAs (miRBase v.10.1). The Spearman correlation between cloning and microarray data is 0.7 corresponding to a p-value <3.9e-28 (
Discussion
The combination of cloning procedures and computational tools used in this study led to the identification of a large fraction of miRNA expressed during B-cell differentiation. These included 75 previously unreported miRNAs, as well as a potentially distinct class of short-RNAs not fulfilling current criteria for miRNAs. These findings have general implications for the understanding of the total miRNA content of the human genome as well as for future studies on the role of miRNAs in B-cell differentiation, function and lymphomagenesis.
The discovery of 75 previously unreported miRNAs expressed in normal and/or malignant B cells is in contrast with a previous study that reported the discovery of only 12 new human miRNA (Landgraf et al., 2007) from an analysis of a large panel of different organ systems and cell types and suggested that most miRNAs have already been identified and are ubiquitously expressed (Landgraf et al., 2007). These discordant results and conclusions may be due i) to the higher number of clones per library sequenced in this study (3500 versus 1300 on average in (Landgraf et al., 2007)), which allowed the detection of low-abundance miRNA species and ii) to the criteria applied in the miRNA identification which do not include conservation and allow consideration of repetitive elements (see Supplemental Experimental Procedures in Example 3). Moreover, the relatively lower degree of evolutionary conservation of previously unreported miRNAs may have prevented the cross-species identification of miRNAs using murine libraries (Chen et al., 2004; Neilson et al., 2007). Consistent with these observations, a recent report on short-RNAs in mouse embryonic stem cells discovered Dicer-dependent miRNAs characterized by both low abundance and low degree of conservation (Calabrese et al., 2007). Since 88% of the previously unreported miRNAs have been independently detected by RT-PCR and/or RNA blot analyses, our cloning and computational approach is largely validated.
We note that a fraction of the validated miRNAs display similarity to the 3′-end of post-transcriptionally modified tRNAs, raising the possibility that they may derive from loci with t-RNA homology or by direct processing of t-RNAs. We also identified a large set of candidate miRNAs (101 unreported, to our knowledge, and 27 known) that have been cloned as single occurrences in the B-cell libraries (Table 7). This group of candidate miRNAs has not yet been fully investigated, but nevertheless they may include bona fide miRNAs because 3 out of 3 tested were detectable by RNA blot or RT-PCR analyses. Thus, our data in B cells suggest that a large number of low-abundance, recently evolved, tissue-specific miRNAs remain to be discovered.
Two categories of short-RNAs were identified that could not be annotated as bona fide miRNAs. The first category is represented by short-RNAs which display all features required by the computational pipeline to be defined as candidate miRNAs, but nevertheless have an atypical length (<17 nt or >28 nt; 75 candidate miRNAs). Sequences belonging to this first category may include bona fide miRNAs since 2 out of 2 tested were detectable by RT-PCR. The second category is represented by those short-RNAs for which classic pre-miRNA structures could not be identified in the genome and no similarity to other non-coding RNA was found in the available databases. These short-RNAs may either be miRNA for which RNA secondary structure prediction algorithms failed to predict the correct hairpin structure or may represent new miRNA species of presently unknown mechanism of generation or other not yet described types of short-RNAs.
Finally, this analysis led to the discovery of short-RNAs that could not be accurately mapped to the genome. Considering that a fraction of these RNAs were cloned multiple times and showed a stage-specific behavior, we suggest that such short-RNAs do actually exist and that the lack of a match to the human genome may be due to polymorphisms, editing and other post-transcriptional modifications or to an incomplete or inaccurate sequencing of the corresponding genomic regions.
The specificity in mature miRNA expression appears to be regulated at the transcriptional as well as at the post-transcriptional, i.e. pre-miRNA processing, level. Indeed, the accumulation of pre-miRNA in absence of a mature miRNA can occur in a cell type-restricted manner, suggesting the presence of a mechanism of regulation at the pre-miRNA processing step. Both regulatory mechanisms may act during normal differentiation and may also be dysregulated during transformation as a consequence of genetic or epigenetic alterations (Lee et al., 2007; Michael et al., 2003; Thomson et al., 2006). Indeed, miRNAs CU-1137 and CU-1368 represent examples of transcriptional activation and post-transcriptional silencing associated with malignant transformation, respectively.
The stage-specific expression of various miRNAs strongly suggests highly specialized regulatory functions in B-cell biology. The role of miRNAs that show cell type-specific functions in lymphocytes has just begun to be elucidated (Dorsett et al., 2008; Li et al., 2007; Rodriguez et al., 2007; Teng et al., 2008; That et al., 2007; Xiao et al., 2007). The miRNAs specifically associated with GC or non-GC B cells by either cloning or miRNA expression profiling have not been previously reported in B-cell differentiation with the exception of miR-150 (Xiao et al., 2007). For example the miR-199 and miR-125 families as well as miR-138 show a distinct expression in GC B cells although none of these miRNAs has been investigated for a role in this cell compartment. The extent of post-transcriptional regulation added by miRNAs will be fully uncovered only in the context of the complex network of cellular interactions (Basso et al., 2005), which will require the integration of large scale gene and miRNA expression data.
miRNA expression can be affected by malignant transformation. For instance, the miR-17-92 cluster, previously reported as a potential oncogene (He et al., 2005), was found over-expressed in Ramos cell line compared to GC B cells. Moreover, several miRNAs (i.e. CU-1137, CU-1148) show expression in Ramos cells and in several additional BL cell lines, but not in their normal GC counterpart. Vice versa, as observed for the miR-199 family, the expression of some miRNAs is lost in the tumor cells. The data herein represents a useful basis to investigate whether lymphoma-associated chromosomal lesions affect genomic regions associated with miRNA expression.
Finally, the differences in miRNA expression profile between GC and non-GC B cells resembled those observed by expression profiling of coding genes (Klein et al., 2003), consistent with the previous observation that miRNA profiling may be equally or more informative in discriminating tumor phenotypes (Calin et al., 2005; Lu et al., 2005). This suggests that miRNA expression profiling, especially if including new B-cell specific miRNAs, may be useful in the differential diagnosis of lymphoid malignancies.
The expanded B-cell miRNome described here represents a resource which can be used to identify miRNAs expressed during the GC transit as well as specific differences in miRNA expression in normal versus lymphoma cells, and which can guide studies to unveil the function of miRNAs in normal B cell development and lymphomagenesis.
Experimental Procedures
Generation of Short-RNA Libraries.
Purification of naïve, memory and GC B cells was performed as previously reported (Klein et al., 2003) using magnetic cell sorting of mononucleated cells obtained from human tonsils. Total RNA was purified using the Trizol Reagent (Invitrogen) following the manufacturer's indications. The short-RNA libraries were generated using an established protocol described in detail in (Lau et al., 2001). Briefly, total RNA was separated on 15% polyacrylamide gel and the fragment corresponding to 15-30 nucleotides length was excised. The purified small RNAs were linked to adaptor oligonucleotides and gel purified. Upon adaptor ligation, RNA was reverse transcribed and cDNA was PCR amplified and cloned into pCR2.1-TOPO vector (Invitrogen). Sequencing was performed on colony PCR amplicons.
Computational Identification of Precursor and Mature miRNAs.
The bioinformatics miRNA analysis pipeline (
Orthology Analysis.
The identification of putative orthologous sequences of known and predicted precursor and mature human miRNAs in chimp (panTro2), monkey (rheMac2), dog (canFam2), mouse (mm8) and rat (rn4) was performed using UCSC-provided Blastz (Schwartz et al., 2003) pairwise alignments between human and target species. The details are reported below.
miRNA Expression Profiling.
The miRNA expression profiles were generated using the Human miRNA Microarray kit (Agilent Technologies) that allows detection of 723 known human (miRBase v.10.1) and 76 human viral miRNAs, following the manufacturer's indications. Analysis of raw data was performed using the Feature Extraction Software 9.5.3.1 (Agilent Technologies). The dendrograms (
RT-PCR Analysis.
Small RNA fractions were purified using the Trizol Reagent (Invitrogen) and the PureLink miRNA Isolation Kit (Invitrogen), following the manufacturer's indications. RT-PCR was performed as previously described (Sharbati-Tehrani et al., 2008). Briefly, miRNA sequences were reverse-transcribed from 50 ng short-RNA using Superscript III First Strand Synthesis Kit (Invitrogen), in the presence of 0.2 μM RTFS primer (miRNA-specific primers, see Table 14). 1/10th of the cDNA volume was then used as template for 34 cycles of PCR amplification in the presence of 4 nM SS primer (miRNA-specific primers, see Table 14) and 0.4 μM each of MPF and MPR universal primers (Table 14). PCR products were separated on 12% non-denaturing polyacrylamide gel, detected by SybrGold (1:10,000 dilution; Invitrogen) and visualized under UV light.
CCCGGGTTTCGGCA
CCA
GGGTTTCG
GCCGGGTACTTTCG
TATTTT
GGGTACTTT
GCTAAGGAAGTCCT
GTGCTCAGTTTT
GC
AAGGAAGTCCTGT
TATCAATGATGCTT
CTGAGA
CAATGATGCTT
TCGATTCCCGGCCC
ATGCACCA
ATTCCCGGCCCAT
AGAAAGGCCGAATT
TTA
CGG
AAGGCCG
TGGTGTGGTCTGTT
GTTTT
AG
TGTGGTCTG
CCCCCCACTGCTAA
ATTTGACTGGCTT
CCCACTGCTAAATTTG
TCCCCGCACCTCCA
CCA
CCGCACCT
GAGAGCGCTCGGTT
TTT
AGCGCT
ATCCCACTCCTGAC
ACCA
CCACTCCTG
GGCGTGATTCATAC
CTTTT
TG
GTGATTCAT
AGGGTGTGCGTGTT
TTT
CGC
GTGTGCGT
AACCGAGCGTCCAA
GCTCTTTCCATTTT
CGAGCGTCCAAGCTCT
TCCCCGACACCTCC
ACCA
CCGACACCT
GCCCGCATCCTCCA
CCA
CGCATCCT
ATGTGGTGGCTTAC
TTTT
GC
TGGTGGCTT
TCCCCGGCACTTCC
ACCA
CCGGCACTT
TCACCCCATAAACA
CCA
CCCCATAA
TTCCCCGACGGGGA
GCCA
CCCGACGGG
TGTGCTCCGGAGTT
ACCTCGTTT
GCTCCGGAGTTA
TCACGTCGGGGTCA
CCA
CGTCGGGG
AGTCCCATCTGGGT
CGCCA
CCCATCTGGG
TCCCCGTACGGGCC
ACCA
CCGTACGGG
GTCCCTTCGTGGTC
GCCA
CCTTCGTGG
GTCAGGATGGCCGA
GCGGTCT
AGGATGGCCGAG
AGGGGGGTAAAAAA
AAA
GGGGTAAA
CCCACCCAGGGACG
CCA
ACCCAGGG
TCCCCGGCACCTCC
ACCA
CCGGCACCT
GAGGGGGACCAAAA
AAAA
GG
GGGGA
TACCGAGCCTGGTG
ATAGC
CGAGCCTGGT
TCGATTCCCGGCCA
ATGCACCA
TG
ATTCCCGGC
GAGCCATGATGATA
CCACTGAGC
CCATGATGATACCA
TAACGGCCGCGGTA
CCC
CGGCCGCG
GCAGCGCCAGCCTC
CCGCCCTAC
GCGCCAGCCTCCCG
CGTCCATGATGTTC
CGCAA
CCATGATGTT
AGCAGTGATGTCCT
GAAAATTCTGAAG
TTT
AGTGATGTCCTGA
AAAGGACCTGGCGG
TGCTTC
GGACCTGGCGG
ATCCCGGACGAGCC
CCCA
CCGGACGAG
TCCTCACACGGGGC
ACCA
TCACACGGG
ATCCCACTTCTGAC
ACCA
CCACTTCTG
ACCCCACTATGCTT
AGCCCT
CCACTATGCTT
TGTATTGTGAGACA
TTC
ATTGTGAG
TCTCGGTGGAACCT
CCA
CGGTGGAA
ATCCCCAGCACCTC
CACCA
CCCAGCACCT
AGAACACTACGAGC
CACA
ACACTACGA
ACCCCACTTCTGGT
ACCA
CCACTTC
CGTTCGCGCTTTCC
CCTG
AG
TCGCG
GACGAGGTGGCCGA
GTGG
GAGGTGGCC
TCCCCGGCATCTCC
ACCA
CCGGCATCT
CTGATTGCTCCTAT
CTGATT
ATTGCTCCTAT
TCTAGAGGAGCCTG
TTCTGTA
AGAGGAGCCTGT
TCGGGTGCGAGAGG
TCCCGGGT
CC
GGTGCGAGA
ATAGGTTTGGTCCT
AGCCTTTCT
GGTTTGGTCCTAGC
TCGATTCCCGGTCA
GGGAACCA
ATTCCCGGTCAGG
TCCTCGTTAGTATA
GTGGTGAGTATCCC
TCGTTAGTATAGTGGT
TCCCTGGTGGTCTA
GTGGTTAGGATTCG
CTGGTGGTCTAGTGGT
TAAGTGTTTGTGGG
TTA
A
GTGTT
GCATTGGTGGTTCA
GTGGTAGA
TTGGTGGTTCAGT
TGGTTATCACGTTC
GCC
T
TTATC
CCCTGCTCGCTGCG
CCA
TGCTCGCT
TTCTCACTACTGCA
CTTGACTA
TCACTACTGCACT
CTCCTGGCTGGCTC
GCCA
CTGGCTGGC
CTCCCACTGCTTCA
CTTGACTAGC
CCACTGCTTCACTTG
CTGCTGTGATGACA
TTC
CTGTGATG
TCCTGCCGCGGTCG
CCA
TGCCGCGG
GCGGGTGATGCGAA
CTGGAGTCTGAGC
GGTGATGCGAACTGGA
CCCCCACAACCGCG
CTTGACTAGC
CCACAACCGCGCTTG
TAGGGGTATGATTC
TCGCT
GGGTATGATT
GGCTGGTCCGAGTG
CAGTGGTGTTTA
AC
TGGTCCGAGTGCAGTG
GGCTGGTCCGATGG
TAGTGGGTT
TGGTCCGATGGTAG
CCACGAGGAAGAGA
GGTAGC
T
CGAGGAAG
CGGAAGCGTGCTGG
GCCC
AAGCGTGCT
GGAGAGAACGCGGT
CTGAGTGGT
GAGAACGCGGTCTG
ATCCCCAGCATCTC
CACCA
CCCAGCATCT
CCCCCCACTGCTAA
ATTTGACTGGA
CCCACTGCTAAATTTG
RNA Blot.
Total RNA and small RNA fractions were purified using the Trizol Reagent (Invitrogen) and the PureLink miRNA Isolation Kit (Invitrogen), respectively, following the manufacturer's indications. Electrophoresis was performed on 15% denaturing polyacrylamide gel and then RNA was transferred on Duralon UV membrane (Stratagene) using a semidry transfer apparatus. Pre-hybridization and hybridization were performed in 5×SSC, 20 mM Na2HPO4 pH7.2, 7% SDS, 3×Denhardt's Solution. Oligonucleotide probes were [γ-32P]-ATP labeled by polynucleotide kinase (Fermentas). The list of oligonucleotides and their hybridization temperature is reported in Table S8. After over-night hybridization, membranes were washed at the same temperature in 3×SSC, 25 mM NaH2PO4 pH 7.5, 5% SDS, 10×Denhardt's Solution for 15-20′ and in 1× SSC, 1% SDS for 5′. Images were obtained by exposure to phosphoimager cassette and acquisition by Storm 840 Phosphoimager (Molecular Dynamics) and by film exposure for approximately 2 weeks.
Estimation of Library Complexity.
A bootstrap technique was used to estimate the total number of miRNAs expressed in each library and the number of short-RNAs must be sequenced to achieve a complete coverage. Bootstrapping is a statistical technique for estimating properties of an “estimator” by measuring those properties in multiple subsets of the samples (Harrell, 2001; Hinkley, 1997). Specifically, we estimated the distribution of mature miRNAs obtained by random sub-sampling different size short-RNA libraries from each complete library. For each size N=10, 20, . . . Nt, where Nt is the total number of short-RNAs in the library, we randomly sampled 1000 libraries of size N and computed the number r(N) of inferred miRNAs, resulting in a distribution p(r(N)) for which we could compute standard statistical parameters such as average, variance, mode and median. Based on this sampling, we can extrapolate p(r(N)) for increasing values of N to determine at which point it is no longer efficient to use larger values of N to increase miRNA coverage. To achieve this, we fitted the data to the parametric function ƒ(x)=K*(1−e−mx).
Since we include both experimentally confirmed and putative mature miRNAs and since bootstrapping can produce optimistic results we expect that the estimated values constitute an upper boundary on the real library complexity. Based on this analysis, we estimated that the total numbers of mature miRNAs are: 129 (naïve), 154 (memory), 204 (centroblasts) and 189 (Ramos). Thus, the libraries sequenced in this study cover respectively 90.7% (naïve), 88.3% (memory), 85.8% (centroblasts), and 91% (Ramos) of the expressed miRNAs in these cellular phenotypes.
Orthology and Conservation Analysis.
We investigated conservation of known and predicted precursor and mature human miRNA in chimp (panTro2), monkey (rheMac2), dog (canFam2) mouse (mm8) and rat (rn4). We obtained 678 miRNA precursor sequences from miRBase (v.11.0), 666 mature miRNAs and 167 star sequences. In total, we obtained 947 locations for mature and star mirBase sequences. We predicted 388 precursors of which 114 match miRBase precursors and 274 are newly predicted. Categorizing these by their corresponding mature sequences, 255 precursors correspond to mature miRNAs that are not included in the miRBase and 133 precursors are associated with 103 predicted miRNAs that match miRBase miRNAs. Of the 274 newly predicted precursors, 19 associated with 8 mature sequences listed in miRBase database.
miRNA conservation has been repeatedly used to help identify putative miRNA mappings to genomes. To identify putative ortholog miRNAs we relied on UCSC-provided Blastz pairwise alignments between human and target species (Schwartz et al., 2003). We used two related but complementary methods: (1) map the mature human miRNA to its ortholog location as specified by pairwise alignment; and (2) map the precursor of the human miRNA to its ortholog location as specified by pairwise alignment, expanding the human region to include at least 80 bases from both sides of the mature region, and identifying regions in the target that match the sequence of the mature human miRNA.
Method 1 is the simplest but fails to account for alignment inaccuracies and local mutations that may shift the position of the mature sequence in the target species. Method 2 accounts for locally imperfect Blastz mapping, but relies on conservation of larger regions that may not be subject to the same selective pressure as the mature miRNA. Alignment-based mapping of the human mature miRNA to its target were required to have either perfect conservation of the entire mature miRNA sequence or conservation of seeds composed of seven bases starting from the second position of the human mature sequence followed by conservation of 3 bases starting from the 12th, 13th or 14th position as suggested by (Grimson et al., 2007) (Appendix Table 11). We scanned the entire mapped ortholog region for a match to the human mature sequence or to its seed.
miRNA Target Prediction and Analysis.
Target predictions for not previously reported miRNAs were performed by miRanda v1.0 (John et al., 2004) and RNA22 (Miranda et al., 2006) using recommended parameters with the exception of RNA22 energy threshold that was changed from default −25.0 kcal/mol to −20.0 kcal/mol.
In order to investigate the potential effect of miRNAs on the transcriptome, predicted targets were tested for enrichment in genes down-regulated in the same population over-expressing the tested miRNA. Over-expressed miRNA were selected based on a minimum frequency value >0.08 and a three-fold increase in their cloning frequency comparing CB vs naïve or memory B cell libraries. Genes differentially expressed across normal B cell populations were identified based on intensity fold change greater than 1.5, and p-value under 0.01 according to a non-parametric U test applied to six biological replicates per cell type (gene expression data are available from GEO database; GSE2350).
For each miRNA, using a Fisher exact test, we compared the numbers of down- and up-regulated predicted targets to down- and up-regulated genes that are not predicted targets. Setting a p-value threshold of 0.01, targets of most GC-specific miRNAs were significantly down regulated in CB. Conversely, targets of naïve- and memory-specific miRNAs were not significantly differentially regulated. Therefore the analysis was focused on targets of the 15 GC-specific miRNAs. Predicted targets of 8 out of 15 miRNAs showed significant enrichment (p-value <0.001) in genes down-regulated in GC compared to naïve B cells and 2 of them showed enrichment for genes down-regulated in a control population (memory compared to naïve) (Table 13). We can conclude that targets of GC-specific miRNAs are significantly more likely to be down regulated in CB than in naïve B cells with p<0.05 according to a Fisher exact test. Moreover, down-regulation p-values in CB were systematically lower than in memory (
Correlation Between Cloning and Microarray miRNA Profiling.
In order to compare cloning and microarray data, we focused on the 89 miRNAs for which both types of data were available. A significant correlation (p-value ≦3.9e-28) was shown between cloning and miRNA microarray data as measured by Spearman correlation. The corresponding scatter plot is shown in
Immunoprecipitation.
Immunoprecipitations were performed from Ramos cells grown in IMDM, 10% fetal bovine serum, 1% Penicillin/Streptomycin. 1-2×10^8 cells were collected and resuspended in 1 ml lysis buffer (10 mM Tris pH 7.5, 2 mM MgCl2, 10 mM KCl, 2.5 mM DTT, 1× protease inhibitors, 40 U/ul Ambion Superase-IN). Lysate supernatant was mixed with 500 ul ATP depletion mix (450 mM KCl, 100 mM glucose, 0.5 U/ul Sigma-Aldrich hexokinase). Cleared supernatant was divided equally between paramagnetic protein G beads (New England Biolabs) bound to either a monoclonal rat antibody raised against human Ago2 protein (Rudel et al., 2008) or total purified rat IgG (Sigma-Aldrich). Beads were incubated with lysate under rotation for 2 hours at 4° C., then washed three times with ice-cold lysis buffer and collected in Trizol (Invitrogen) for RNA extraction. RNA from three sequential immunoprecipitations was pooled and 1/10th of yield was used for reverse transcription of each miRNA species using Superscript III First Strand Synthesis Kit (Invitrogen), in the presence of 0.2 μM RTFS primer (miRNA-specific primers, see Table S8). cDNA was also generated from reverse transcription in the presence of random hexamers to test expression of 5s rRNA. 1/10th of the cDNA volume was used as template for SYBR (Applied Biosystems) qPCR amplification in the presence of 4 nM SS primer (miRNA-specific primers, see Table 14) and 0.4 μM each of MPF and MPR universal or 0.4 μM each of 5sRNA primers (Table 14). Each qPCR reaction was performed in triplicate. The tested miRNA were selected based on the availability of optimized qRT-PCR conditions among the ones detectable both by RNA blot and RT-PCR.
Accession Numbers.
The miRNA array profiles data are available from the GEO repository (GSE15144).
This application is a Continuation-In-Part of International Patent Application No. PCT/US2008/070082, filed Jul. 15, 2008, which claims priority of U.S. Provisional Patent Application No. 60/950,474, filed Jul. 18, 2007, and of U.S. Provisional Patent Application No. 61/020,625, filed Jan. 11, 2008 each of which is incorporated herewith in its entirety.
The work described herein was supported in whole, or in part, by National Cancer Institute Grant No. R01-CA109755 “Genetic Network Interference with Combinatorial Phenotypes”, and National Institute of Allergy and Infectious Diseases Grant No. R01 AI066116 “Regulatory Modules in Normal and Transformed b-Cell”. Thus, the United States Government has certain rights to the invention.
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| Number | Date | Country | |
|---|---|---|---|
| 20100197772 A1 | Aug 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60950474 | Jul 2007 | US | |
| 61020625 | Jan 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2008/070082 | Jul 2008 | US |
| Child | 12688680 | US |
