Patent Application
20160319364
The invention provides methods and reagents for amplifying and detecting microRNAs (miRNAs). More particularly, the invention provides methods and reagents for amplifying, measuring, and identifying miRNAs from limited tissue samples or cell samples. In addition, the invention provides bioinformatical methods for miRNA target identification by analyzing correlations between expression of miRNAs and their candidate target mRNAs. Such methods are useful for discovering miRNA cancer biomarkers and for cancer diagnostics.
miRNAs are short (˜22 nucleotides) non-coding RNAs involved in post-transcriptional silencing of target genes. In animals, miRNAs control target gene expression both by inhibiting translation and by marking their target mRNAs for degradation. Although much less common, recent reports indicate that miRNAs can also stimulate target gene expression (Buchan et al., 2007, Science 318: 1877-8; Vasudevan et al., 2007, Science 318: 1931-34; Vasudevan et al., 2007, Cell: 128:1105-118; Bhattacharyya et al., 2007, Cell: 128: 1105-118; Wu et al., 2008, Mol Cell 29: 1-7). The mechanism of miRNA action is through binding to the 3′ untranslated regions (UTRs) of target mRNAs, with varying degrees of sequence complementarity (Bartel, 2004, Cell 116: 281). miRNAs regulate genes associated with development, differentiation, proliferation, apoptosis and stress response, but have also been implicated in multiple cancers, for example: miR-15 and miR-16 in B-cell chronic lymphocytic leukemias (Calin et al., 2002, Proc Natl Acad Sci USA. 99:15524-9; Calin et al., 2004, Proc Natl Acad Sci USA. 101:11755-60); miR-143 and miR-145 in colorectal cancer (Michael et al., 2003, Mol Cancer Res. 1:882-91); miR-125b, miR-145, miR-21, miR-155 and miR-17-5p in breast cancer (Iorio et al., 2005, Cancer Res. 65:7065-70; Hossain et al., 2006, Mol Cell Biol. 26:8191-201); and miR-21 in glioblastoma (Chan et al., 2005, Cancer Res. 65:6029-33). Several miRNAs have been mapped to cancer-associated genomic regions (Calin et al., 2004, Proc Natl Acad Sci USA. 101:2999-3004). The expression of the let-7 miRNA has been correlated with prognosis in lung cancer (Takamizawa et al., 2004, Cancer Res. 64:3753-6) and found to regulate RAS in the same tumor (Johnson et al., 2005, Cell. 120:635-47). Very recently, mir-10b has been shown to contribute to metastasis in breast cancer (Ma et al., 2007, Nature. 449:682-88). This evidence indicates that miRNAs likely affect the development and maintenance of a variety of cancers. Although many miRNAs have been implicated in regulating cancers, very few of their target genes, and hence their downstream mode of action, have been identified.
Tumors often are heterogeneous in cell content, with the true tumor cell mass interspersed with or in close proximity to non-tumor cells. To determine miRNA levels that reflect the status of the tumor cells, measurements derived from stromal and other contaminating cells present in the tumor need to be excluded. This can be achieved by isolating the tumor cells using, inter alia, laser capture-microdissection (LCM) from thin sections of the tumor mass. Although this process achieves isolation of a pure population of the desired cell type(s), the number of cells obtained is limited, and consequently, yields of RNA are low. There is a need in the art, accordingly, for methods permitting miRNA expression detection and profiling from very limited amounts of starting RNA such as obtained from cells isolated by LCM.
The association of miRNA molecules with certain cancers illustrates the need for using the expression levels of these molecules as biomarkers for cancer diagnostics. There is an equally important need to identify mRNA targets of said miRNAs, in order to identify the affected cellular genes and processes involved in tumor initiation, progression and metastasis.
The invention provides methods for amplification and measurement of levels of a plurality of miRNAs in a biological sample, preferably comprising all or a substantial portion thereof of miRNAs in a sample. In addition, the invention provides methods for assessing miRNA profile complexity, preferably in limited amounts of a biological cell or tissue samples and most particularly, in limited amounts of tumor samples. The disclosed methods include assessment of miRNA levels and related mRNA levels, to identify miRNA-specific target mRNAs. One application of said methods is thus to identify cancer biomarkers among both miRNA and target genes.
In the practice of the methods of this invention, oligonucleotide primers are ligated exclusively to miRNAs in RNA extracts from a cell or tissue sample, followed by a series of amplification steps to generate multiple miRNA copies (a non-limiting, exemplary illustration of said methods is shown in
In certain embodiments, the invention provides methods for measuring differential expression of miRNAs between control samples and experimental samples. miRNA levels in experimental samples, such as diseased or cancerous tissue sections, are measured and compared to miRNA levels present in control or non-diseased tissues, most preferably wherein the control or non-diseased tissue is from the same tissue source (e.g., normal colon epithelia vs. colon cancer). miRNA species whose levels have the greatest difference between experimental and control tissues are designated as biomarker candidates.
Because miRNAs function by regulating gene expression post-transcriptionally, identification of the target mRNAs complementary to miRNA biomarkers assists in the elucidation of the molecular basis of malignancy and/or disease pathology. This aspect of the invention also identifies additional cancer biomarkers, and particularly biomarkers that can be detected using additional methodologies, including inter alia antibody detection of mRNA gene product(s). Thus, the invention provides methods for identifying downstream mRNA targets of miRNA inactivation that are associated with a cancer phenotype. Candidate miRNA target mRNAs are defined by having sequence complementarity, particularly in their 3′ untranslated region (3′-UTR), to a particular miRNA (as illustrated in
In addition to these methods, the invention provides a particular miRNA species, miR-29c, as a cancer biomarker for nasopharyngeal carcinoma. The invention provides a plurality of downstream mRNA targets of miR-29c, including several genes expressing extracellular matrix proteins (ECMs). The measurement of miR-29c and/or its target mRNAs in patient samples thus comprises a cancer diagnostic reagent. As demonstrated, by the experimental evidence disclosed herein, miR-29c downregulates expression of multiple genes encoding ECM components or genes related to ECM when an miR-29c-encoding construct is artificially transfected into cells in culture. The ECM related genes whose expression is downregulated by miR-29c include Collagens 1A2 (GenBank Accession No. NM_000089), 3A1 (NM_000090), 4A1 (NM_001845), 15A1 (NM_001855), Laminin-γ1 (NM_002293) and Fibrillin1. miR-29c also downregulates Thymine-DNA glycosylase (TDG) (NM_003211) and FUSIP1 (NM_006625, NM_054016) (shown in
Advantages of the practice of this invention include, inter alia, that it permits measurement of miRNA expression levels in enriched tumor cell populations from patient biopsies isolated by methods such as LCM, from limited tumor cell sources that, prior to this invention, yielded insufficient total RNA for miRNA expression profiling.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.
These and other objects and features of this invention will be better understood from the following detailed description taken in conjunction with the drawing wherein:
The sequence identifiers for the sequences disclosed in
Collagen 3A1 homo sapiens upstream sequence (SEQ ID NO: 18) and downstream sequence (SEQ ID NO: 19); Collagen 3A1 Pan trogolodytes upstream sequence (SEQ ID NO: 20) and downstream sequence (SEQ ID NO: 21); Collagen 3A1 Mus musculus upstream sequence (SEQ ID NO: 22) and downstream sequence (SEQ ID NO: 23); Collagen 3A1 Rattus norvegicus upstream sequence (SEQ ID NO: 24) and downstream sequence (SEQ ID NO: 25); Collagen 3A1 Canis familiaris upstream sequence (SEQ ID NO: 26) and downstream sequence (SEQ ID NO: 27); Collagen 3A1 Gorilla gorilla upstream sequence (SEQ ID NO: 28) and downstream sequence (SEQ ID NO: 29).
Collagen 4A1 homo sapiens upstream sequence (SEQ ID NO: 30) and downstream sequence (SEQ ID NO: 31); Collagen 4A1 Pan trogolodytes upstream sequence (SEQ ID NO: 32) and downstream sequence (SEQ ID NO: 33); Collagen 4A1 Mus musculus upstream sequence (SEQ ID NO: 34) and downstream sequence (SEQ ID NO: 35); Collagen 4A1 Rattus norvegicus upstream sequence (SEQ ID NO: 36) and downstream sequence (SEQ ID NO: 37); Collagen 4A1 Canis familiaris upstream sequence (SEQ ID NO: 38) and downstream sequence (SEQ ID NO: 39); Collagen 4A1 Gorilla gorilla upstream sequence (SEQ ID NO: 40) and downstream sequence (SEQ ID NO: 41).
Fibrillin 1 homo sapiens upstream sequence (SEQ ID NO: 42) and downstream sequence (SEQ ID NO: 43); Fibrillin 1 Pan trogolodytes downstream sequence (SEQ ID NO: 44); Fibrillin 1 Mus musculus upstream sequence (SEQ ID NO: 45) and downstream sequence (SEQ ID NO: 46); Fibrillin 1 Rattus norvegicus upstream sequence (SEQ ID NO: 47) and downstream sequence (SEQ ID NO: 48); Fibrillin 1 Canis familiaris upstream sequence (SEQ ID NO: 49) and downstream sequence (SEQ ID NO: 50); Fibrillin 1 Gorilla gorilla upstream sequence (SEQ ID NO: 51) and downstream sequence (SEQ ID NO: 52); Fibrillin 1 Fugu rubripes upstream sequence (SEQ ID NO: 53) and downstream sequence (SEQ ID NO: 54).
Thymine DNA Glycosylase homo sapiens upstream sequence (SEQ ID NO: 55), middle sequence (SEQ ID NO: 56) and downstream sequence (SEQ ID NO: 57); Thymine DNA Glycosylase Pan trogolodytes upstream sequence (SEQ ID NO: 58), middle sequence (SEQ ID NO: 59) and downstream sequence (SEQ ID NO: 60); Thymine DNA Glycosylase Mus musculus upstream sequence (SEQ ID NO: 61), middle sequence (SEQ ID NO: 62) and downstream sequence (SEQ ID NO: 63); Thymine DNA Glycosylase Rattus norvegicus upstream sequence (SEQ ID NO: 64), middle sequence (SEQ ID NO: 65) and downstream sequence (SEQ ID NO: 66); Thymine DNA Glycosylase Canis familiaris upstream sequence (SEQ ID NO: 67), middle sequence (SEQ ID NO: 68) and downstream sequence (SEQ ID NO: 69); Thymine DNA Glycosylase Gorilla gorilla upstream sequence (SEQ ID NO: 70).
The sequences disclosed in the figure include miR-29c 5′ UAGCACCAUUUGAAAUCGGU 3′ (SEQ ID NO: 1). The same miR-29c sequence is also represented throughout the
This invention provides methods and reagents for measuring miRNA expression in a biological sample, preferably a cell or tissue sample and even more preferably a tumor sample, and particularly when the amounts of such samples are limited in size and/or the number of cells. The term “limited” as used herein refers preferably to a range of approximately 1000-10,000 cells. In a preferred embodiment, cell numbers range from approximately 1000-10,000 cells, or alternatively 1000-5000 cells, in certain alternative embodiments approximately 1000 cells or in certain samples from about 500-1000 cells, in yet other samples 10-500 cells or at a minimum at least one cell.
In turn, the methods disclosed herein permit miRNA expression from minute amounts of starting RNA to be identified. The term “minute” as used herein refers to very low amounts of total RNA. In a preferred embodiment, starting RNA will comprise about 30-100 ng of RNA, preferably 50-90 ng, and more preferably 75-85 ng. The invention thus provides methods for assessing differential expression of miRNA species between biological samples, particularly cell or tissue samples and even more preferably tumor samples, and control, preferably non-tumor samples, wherein the tumor samples are enriched for tumor cell content as described herein. The invention also provides methods for identifying one or a plurality of miRNA-complementary target mRNAs from cellular genes whose expression is modulated (upregulated or downregulated) by expression of one or a plurality of miRNA species. The inventive methods are useful for the identification of disease biomarkers, particularly cancer biomarkers.
The term “biomarker” as used herein refers to miRNA, mRNA or protein species that exhibit differential expression between biological samples, preferably patient samples and more preferably cancer patient samples, when compared with control patient samples. The term “patient sample” as used herein refers to a cell or tissue sample obtained from a patient (such as a biopsy) or cells collected from in vitro cultured samples; the term can also encompass experimentally derived cell samples. In a preferred embodiment, patient samples are laser-microdissected, inter alia from frozen tissue sections. Cells from patient samples can be used directly after isolation from biopsy material or can be in vitro propagated.
As used herein, the terms “experimental sample” and “biological sample” refer preferably to a diseased or cancerous tissue sample including specifically cell culture samples and experimentally-derived samples. As used herein, the term “control” sample refers to tissue that is normal or pathology-free in appearance and may be harvested from the same patient or a different patient, most preferably being from the same tissue type as the disease or experimental sample (e.g., normal colon tissue vs. colon cancer) and most preferably otherwise processed as is an experimental, biological or patient sample. The term “tumor” refers to a tissue sample or cells that exhibit a cancerous morphology, express cancer markers, or appear abnormal, or that have been removed from a patient having a clinical diagnosis of cancer. A tumorigenic tissue is not limited to any specific stage of cancer or cancer type, an expressly includes dysplasia, anaplasia and precancerous lesions such as inter alia adenoma. As used herein, the term “disease” or “diseased” refers to any abnormal pathologies, including but not limited to cancer. As used herein, the term “aberrant” refers to abnormal or altered.
As designated herein, miRNA targets are mRNA transcripts that are regulated by miRNA. Regulation of target mRNA can include but is not limited to binding or any sequence-specific interaction between an miRNA and its target mRNA, and includes but it not limited to decreasing stability of the mRNA, or decreasing mRNA translation, or increasing mRNA degradation.
The practice of this invention can involve procedures well-known in the art, including for example nucleotide sequence amplification, such as polymerase chain reaction (PCR) and modifications thereof (including for example reverse transcription (RT)-PCR, and stem-loop PCR), as well as reverse transcription and in vitro transcription. Generally these methods utilize one or a pair of oligonucleotide primers having sequence complimentary to sequences 5′ and 3′ to the sequence of interest, and in the use of these primers they are hybridized to a nucleotide sequence and extended during the practice of PCR amplification using DNA polymerase (preferably using a thermal-stable polymerase such as Taq polymerase). RT-PCR may be performed on miRNA or mRNA with a specific 5′ primer or random primers and appropriate reverse transcription enzymes such as avian (AMV-RT) or murine (MMLV-RT) reverse transcriptase enzymes.
The term “complimentary” as used herein refers to nucleotide sequences in which the bases of a first oligonucleotide or polynucleotide chain are able to form base pairs with a sequence of bases on another oligonucleotide or polynucleotide chain. The terms “sense” and “antisense” refer to complimentary strands of a nucleotide sequence, where the sense strand or coding strand has the same polarity as an mRNA transcript and the antisense strand or anticoding strand is the coding strand's compliment. The antisense strand is also referred to as the anticoding strand.
The term “hybridization” as used herein refers to binding or interaction of complementary nucleotide strands, particularly wherein the complementary bases in the two chains form intermolecular hydrogen bonds between the bases (known in the art as “basepairing”). Hybridization need not be 100% complete base pair matching, meaning some of the bases in a given set of sequences need not be complimentary, provided that enough of the bases are complimentary to permit interaction or annealing of the two strands under the conditions specified, including temperature and salt concentration. In certain embodiments of the invention, hybridization occurs between miRNAs and their target mRNAs, which is often imperfect (e.g. less than 100% complimentary base pairing). miRNAs inhibit translation of target mRNAs by binding to target sequences with which they share at least partial complementarity, wherein said target sequences are most often located within the 3′ untranslated region (UTR) of these target mRNAs. It will be recognized that this is not always a simple function of calculating purported or proposed specificities, since secondary structures (stem-and-loop structures, for example) can affect the stability or accessibility of miRNA/mRNA hybridization. Accordingly, hybridization is most accurately measured by detecting decreased expression of a target mRNA in a cell expressing the complementary miRNA; these methods for detecting intracellular hybridization are also specific for functional miRNA::mRNA hybridization events. Conversely, hybridization between a capture sequence and its corresponding probe will typically have near-perfect to perfect (complete) base pairing (i.e. the sequence experiences extensive complimentary base pairing for a particular sequence or portion of a transcript).
The term “sense targets” as used herein refers to sense strands of miRNA containing a capture sequence. The sense targets are generated by the methods of the invention as disclosed herein. Sense targets can be detected and identified using antisense (i.e., complementary) RNA. In a preferred embodiment, antisense miRNAs are bound to a microarray that is used to detect such sense targets.
The term “capture sequence” as used herein refers to any nucleotide sequence used to hybridize with a detection probe. In a preferred embodiment, the capture sequence is SEQ ID NO: 71. TTC TCG TGT TCC GTT TGT ACT CTA AGG TGG A. This sequence is used in the methods of the invention to identify miRNAs amplified from a sample, which were bound to probe miRNAs affixed to a microarray. In a second hybridization step, a fluorophore-labeled detection probe, with oligonucleotide sequence complementary to the capture sequence, was used to detect those sample miRNAs that bound to the microarray.
The terms “secondary detection probe” or “secondary hybridization” refer to the use of a second hybridization step in a microarray or other hybridization-based analysis. In a preferred embodiment, the capture sequence in amplified miRNAs bound to the microarray by a primary hybridization step is used to hybridize to a complementary oligonucleotide that is linked to a fluorophore, most preferably using fluorescent labels that have excitation and emission wavelengths adapted for detection using commercially-available instruments. Examples of fluorescent labels useful in the practice of the invention include CY3 3DNA™ (Genisphere, PA, USA), phycoerythrin (PE), fluorescein isothiocyanate (FITC), rhodamine (RH), Texas Red (TX), Cy3, Hoechst 33258, and 4′,6-diamidino-2-phenylindole (DAPI). The fluorophore complex in particular permits detection of miRNA by automated microarray scanners.
The term “inversely proportional” as used herein refers to the comparison of expression levels of miRNAs and mRNAs between tissue samples or groups of similar samples. For example, where miRNA expression levels are low in a cancer sample, the methods of the invention identify high miRNA expression in control samples. This differential expression analysis permits identification of potential cancer markers. In a preferred embodiment, the invention identifies mRNAs that are expressed at levels inversely proportional to regulatory miRNAs. For example, where miRNAs are expressed at high levels in a cancer tissue, the methods identify mRNAs that are expressed at low levels in the cancer tissue, since the miRNAs affect mRNA expression in the cancer cell.
The terms “differential analysis” and “differentially expressed” as used herein may refer to, but are not limited to differences in expression levels for miRNAs and/or mRNAs between control and experimental samples. Alternatively, as described above, differential analysis may also include comparisons of expression between miRNAs and potential target mRNAs within the same tissue sample or different tissue samples. In addition, the terms as used herein may refer to the expression of miRNA at greater or lesser amounts in an experimental tissue/experimental cell sample than miRNA expression in a control cell/control tissue sample. The control sample can be from healthy tissue from the same patient or a different patient. Expression of miRNAs may occur in a tissues sample where typically expression does not occur, or expression may occur at levels greater than or less than typically found in a particular cell or tissue type. An example of such differential expression is demonstrated herein for miR-29c in nasopharyngeal carcinoma, as discussed more fully below.
The term “miRNA specific primers” as used herein refers to 3′ and 5′ primers that link to miRNA and permit miRNA amplification. Primers for amplifying miRNA are commercially available and techniques are known in the art. (see, for example, Lau et al., 2001, Science. 294:858-62). In use, primers are ligated to all single-stranded RNA species with a free 3′OH and a 5′ monophosphate, which includes all miRNAs (and specifically excludes eukaryotic mRNA).
As used herein, the terms “microarray,” “bioarray,” “biochip” and “biochip array” refer to an ordered spatial arrangement of immobilized biomolecular probes arrayed on a solid supporting substrate. Preferably, the biomolecular probes are immobilized on the solid supporting substrate.
Gene arrays or microarrays as known in the art are useful in the practice of the methods of this invention. See, for example, DNA M
Gene arrays are complex experimental systems, and their development stemmed from a confluence of various technologies including the Human Genome Project and the development of computational power and bioinformatics applications to DNA sequencing, probe design, and data output analysis (Lockhart et al., 2000, Nature 405: 827-36; Schena et al., 1998, Trends Biotechnol. 16: 301-6; Schadt et al., 2000, J. Cell Biochem. 80: 192-202; Li et al., 2001, Bioinformatics 17: 1067-1076; Wu et al., 2001, Appl. Environ. Microbiol. 67: 5780-90; and Kaderali et al., 2002, Bioinformatics 18: 1340-9). These developments enable one of ordinary skill to produce arrays of polynucleotides from a plurality of different human genes, including polynucleotides complementary to miRNA species.
Two principal array platforms are currently in widespread use, but differ in how the oligonucleotide probes are placed onto the hybridization surface (Lockhart et al., 2000, Id. and Gerhold et al., 1999, Trends Biochem. Sci. 24: 168-73). Schena and Brown pioneered techniques for robotically depositing presynthesized oligonucleotides (typically, PCR-amplified inserts from cDNA clones) onto coated surfaces (Schena et al., 1995, Science 270: 467-70 and Okamoto et al., 2000, Nat. Biotechnol. 18: 438-41). Fodor et al. (1991, Science 251: 767-73) and Lipshutz et al. (1999, Nat. Genet. 21:20-4), on the other hand, utilized photolithographic masking techniques (similar to those used to manufacture silicon chips) to construct polynucleotides one base at a time on preferentially unmasked surfaces containing an oligonucleotide targeted for chain elongation. These two methods generate reproducible probe sets amenable for gene expression profiling and can be used to determine the gene expression profiles of tumor samples when used in accordance with the methods of this invention.
Biochips, as used in the art, encompass substrates containing arrays or microarrays, preferably ordered arrays and most preferably ordered, addressable arrays, of biological molecules that comprise one member of a biological binding pair. Typically, such arrays are oligonucleotide arrays comprising a nucleotide sequence that is complementary to at least one sequence that may be or is expected to be present in a biological sample. As provided herein, the invention comprises useful microarrays for detecting differential miRNA expression in tumor samples, prepared as set forth herein or provided by commercial sources, such as Affymetrix, Inc. (Santa Clara, Calif.), Incyte Inc. (Palo Alto, Calif.) and Research Genetics (Huntsville, Ala.).
In certain embodiments of the diagnostic methods of this invention, said biochip arrays are used to detect differential expression of miRNA or target mRNA species by hybridizing amplification products from experimental and control tissue samples to said array, and detecting hybridization at specific positions on the array having known complementary sequences to specific miRNAs or their mRNA target(s).
In certain other embodiments of the diagnostic methods of this invention, expression of the protein product(s) of mRNA targets of miRNA regulation are detected. In preferred embodiments, protein products are detected using immunological reagents, examples of which include antibodies, most preferably monoclonal antibodies that recognize said differentially-expressed proteins.
For the purposes of this invention, the term “immunological reagents” is intended to encompass antisera and antibodies, particularly monoclonal antibodies, as well as fragments thereof (including F(ab), F(ab)2, F(ab)′ and Fv fragments). Also included in the definition of immunological reagent are chimeric antibodies, humanized antibodies, and recombinantly-produced antibodies and fragments thereof. Immunological methods used in conjunction with the reagents of the invention include direct and indirect (for example, sandwich-type) labeling techniques, immunoaffinity columns, immunomagnetic beads, fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assays (ELISA), and radioimmune assay (MA).
The immunological reagents of the invention are preferably detectably-labeled, most preferably using fluorescent labels that have excitation and emission wavelengths adapted for detection using commercially-available instruments such as and most preferably fluorescence activated cell sorters. Examples of fluorescent labels useful in the practice of the invention include phycoerythrin (PE), fluorescein isothiocyanate (FITC), rhodamine (RH), Texas Red (TX), Cy3, Hoechst 33258, and 4′,6-diamidino-2-phenylindole (DAPI). Such labels can be conjugated to immunological reagents, such as antibodies and most preferably monoclonal antibodies using standard techniques (Maino et al., 1995, Cytometry 20: 127-133).
The methods of this invention detect miRNAs differentially expressed in malignant and normal control tissue. Certain embodiments of the methods of the invention can be used to detect differential miRNA expression in Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC). NPC is a highly metastatic tumor even in the early stage of the disease (Cassisi: Tumors of the pharynx. Thawley et al., eds. Comprehensive Management of Head and Neck Tumors, 1987, Vol 1.: pp 614-683, W. B. Saunders Co., Philadelphia).
Nasopharyngeal carcinoma (NPC) is associated with Epstein-Barr virus (EBV), is found prominently in people in South East Asia, and is highly invasive (Lo et al., 2004, Cancer Cell. 5:423-428). Differential gene expression in NPC relative to normal nasopharyngeal epithelium was examined. Differential expression underlies the properties of this type of tumor, which illustrate the contribution of EBV genes towards immune evasion of tumor cells in this cancer and further implicate DNA repair and nitrosamine metabolism mechanisms in NPC pathogenesis (Sengupta et al., 2006, Cancer Res. 66:7999-8006; Dodd et al., 2006, Cancer Epidemiol Biomarkers Prev. 15:2216-2225).
The invention provides sensitive procedures for amplifying miRNAs from enriched, tumor cell sources, such as laser-microdissected frozen tissue sections (and advantageously assaying a cell or tissue population highly enriched, more preferably very highly enriched, in tumor cells and not stromal or other undesirable cells) and detecting these miRNAs using, for example, microarrays. “Enriched” as used herein indicates that more than approximately 50%, more preferably more than 60%, more than 70%, even more preferably at least 80% and in certain embodiments at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 or 99% of the cells in a sample are of the cells in a sample are of the targeted cell type. The inventive methods have an advantage, inter alia, over traditional methods that require a larger tissue sample that required excision from a patient or alternatively that required that tumor cells from excised tissues be propagated in cell culture, thus relying on the (incomplete) growth advantage of tumor cells over stromal cells, in order to collect sufficient RNA for the subsequent analysis. The differentially-expressed miRNAs detected using the inventive methods thus provided potential tumor markers for malignancy, tumor progression and metastasis.
These inventive methods were able to isolate and amplify minute amounts of miRNA from limited tissue biopsies. For example, needle biopsies typically measure 1 mm diameter by 2 mm length, and experimental samples often comprise one or more ˜20 micron cryosections, which contain very little tissue. These samples generally are not 100% pure tumor cell populations, and thus some samples require laser capture of the tumor component to enrich up to the preferred percentage of epithelial cell type.
In order to identify miRNA cancer biomarkers, two hundred twenty-two (222) human miRNAs were analyzed from thirty-one microdissected NPC samples and ten site-matched normal epithelial tissues. Eight cellular miRNAs were found to be differentially expressed between tumor and normal cells. Two algorithms were used to search for target mRNAs regulated by these miRNAs. {See pictar.bio.nyu.edu/cgi-bin/PicTar_vertebrate.cgi, snf (www.targetscan.org as discussed in Example 4).} One of the miRNA species, miR-29c, was found to be downregulated in NPC and associated with post-transcriptional regulation of multiple extra-cellular matrix protein genes. Increased levels of extracellular matrix proteins, particularly collagens and laminins would be expected to increase the invasiveness and metastasis of many tumor cells. The association between differential expression of miR-29c and extracellular matrix protein expression was confirmed in two epithelial cells in culture, where miR-29c expression was increased artificially, resulting in decreased expression of eight cellular mRNAs, six of which encoded extra-cellular matrix (ECM) proteins. Thus, differential expression of miR-29c miRNA in NPC tissue is consistent with its use as a biomarker, since it had the capacity to contribute to pathogenesis of NPC tumors. These results demonstrated that the methods of this invention were useful for identifying miRNA cancer biomarkers and their downstream mRNA targets.
Once detected, differentially amplified and/or overexpressed miRNAs or mRNAs can be used alone or in combination to assay individual tumor samples and determine a prognosis, particularly a prognosis regarding treatment decisions, most particularly regarding decisions relating to treatment modalities such as chemotherapeutic treatment. Moreover, once differentially-expressed miRNA biomarkers have been identified, potential target mRNAs can be identified by detecting target sequences in said mRNAs, particularly in the 3′ UTR thereof, that are complementary to the capture sequences of the differentially-expressed miRNAs.
Finally, the administration of miRNAs as therapeutics is well known in the art. (See, De Fougerolles, 2008, Human Gene Therapy, 19: 125-32 for a recent review.) Examples 5 and 6 herein illustrate miRNA regulation/modulation of target mRNA expression. Hence miR-29c, miR-29a, miR-29b, miR-34c, miR-34b, miR-212, miR-216 and miR-217, miR-151 or miR-192 and other miRNAs identified by the disclosed methods may be administered as therapeutics for the treatment of cancer, including NPC, and other disorders by methods known in the art.
miRNAs identified according to the methods herein provide targets for therapeutic intervention. miRNAs that are underexpressed, such as miR-29c in tumors such as NPC or in other tumors or other diseases or disorders, can be introduced using conventional nucleic acid formulation and delivery methods. (De Fougerolles, 2008, Human Gene Therapy, 19: 125-3; Akinc et al., 27 Apr. 2008, Nature Biotechnology, advanced online: 1-9). Alternatively, expression of endogenous miR-29c in tumors such as NPC or in other tumors or other diseases or disorders, can be increased, inter alia, using stimulators of miRNA expression. Similarly, expression of miRNAs that are overexpressed can be repressed, using antisense or siRNA methods or by modulating expression using repressors of miRNA expression. The invention also contemplates compounds and pharmaceutical compositions thereof and methods for modulating miRNA expression in a tumor or other tissue to achieve a therapeutic effect.
Embodiments of the methods of this invention comprising the above-mentioned features are intended to fall within the scope of this invention.
The Examples which follow are illustrative of specific embodiments of the invention, and various uses thereof. They set forth for explanatory purposes only, and are not to be taken as limiting the invention.
The methods described in this Example were developed to overcome deficiencies in the art associated with detection and differential expression analysis of miRNAs isolated from limited cell or tissue samples.
Total cellular RNA was isolated from tissue samples including nasopharyngeal carcinoma (NPC) tissue samples. Collection and processing of such samples, including histopathology, laser capture microdissection, and RNA extraction have been described in detail previously (Sengupta et al., 2006, Cancer Res. 66: 7999-8006), the disclosure of which is incorporated by reference herein. Here, a total of thirty-one NPC samples and ten normal nasopharyngeal tissue samples (including six normal tissue samples from non-NPC or biopsy-negative cases and four samples from tumor free nasopharyngeal area of NPC patients) were used. miRNA was amplified from total RNA isolated from laser microdissected/whole tissue sections without any size selection following the procedures disclosed in Lau et al. (2001, Science. 294:858-62, the disclosure of which is incorporated by reference herein) as briefly set forth as follows and illustrated in
Total RNA (˜100 ng) from laser microdissected cells (isolated using Trizol, Invitrogen, Carlsbad, Calif., USA) was used in a ligation reaction where all single stranded RNA species with a 3′ OH were ligated using by RNA ligase I to a “3′ linker” having the sequence:
this oligonucleotide was commercially-available as a miRNA cloning linker from Integrated DNA Technologies (Coralville, Iowa). The reaction was carried out using a modification of the conventional, two-step reaction (where in the first step, ATP was used to adenylate the 5′ end of a nucleic acid and in the second step, the activated adenylated nucleic acid was ligated to the 3′ OH of another nucleic acid). Here, the presence of a 5′ pyrophosphate on the linker moiety permitted the reaction mixture to exclude ATP, with the consequence that the only RNA species in the reaction mixture capable of being ligated to a 3′OH was the linker; this prevented the ligase from nonspecifically ligating unrelated RNA molecules from the tissue sample in the reaction mixture to one another, as well as preventing individual RNA molecules from being circularized. Finally, the presence of the 3′dideoxy-C (ddC) residue in the linker moiety prevented RNA molecules that were ligated to the linker from further participation in the ligation reaction.
The next step for preparing the RNA population for amplification was ligating a linker to the 5′ end of the RNA molecules in the reaction mixture. For this reaction, a “5′ linker” having the sequence:
(wherein uppercase letters designate deoxyribonucleotide residues and lowercase letters are ribonucleic acid residues; commercially-available from Dharmacon RNA Technologies, Lafayette, Colo., USA) was ligated using T4 RNA ligase in the presence of ATP. T4 RNA ligase has a higher ligation efficiency for RNA:RNA ligations, and thus the use of the hybrid DNA:RNA linker inhibited linker self-ligation, and the use of ATP directed ligation to the 5′ monophosphorylated miRNA sequence. Ligation to the 3′ end of the RNA sequences in the reaction mixture was prevented by the presence of the 3′ dideoxy C-containing linker, further directing the ligation reaction to the desired 5′ end of the RNA species, particularly the miRNA species, in the reaction mixture. Full length mRNAs in the reaction mixture were precluded from participating in the 5′ ligation reaction by the presence of the 5′ cap, as were degraded mRNAs by having a 5′ triphosphate which is not a substrate for T4 RNA ligase. Finally, any tRNAs in the mixture are double-stranded at the 5′ end, which inhibits the ligation reaction for those species. rRNAs have extensive secondary structure preventing their ligation and later reverse transcription.
Following linker ligation, the miRNA species were converted to cDNA by reverse transcription using a primer having the sequence: ATT GAT GGT GCC TAC (SEQ ID No: 74) that was complementary to the sequence of the 3′ linker, providing further specificity (Lau et al., 2001, Id.). The resulting cDNA population was amplified by polymerase chain reaction (PCR) using the following primers:
The forward PCR primer sequence contains three regions: the 3′ region is complementary to the 3′ end of the cDNA, while the 5′ region is a T7 RNA polymerase-specific promoter sequence. In between is a sequence complementary to a “capture” sequence identified as SEQ ID NO: 71 (TTC TCG TGT TCC GTT TGT ACT CTA AGG TGG A). PCR was performed using these primers with one initial denaturation of 95° C. for one minute followed by 20 cycles having a profile of denaturation at 95° C. for 20 seconds, primer annealing at 50° C. for one minute, and primer extension at 72° C. for 30 seconds. There was a final extension step at 72° C. for 5 minutes. The reaction mixture contained 10 units of Taq DNA polymerase in its buffer (as supplied by the manufacturer), 0.2 mM dNTPs, 1.5 mM MgCl2, 1 μM primers and the reverse transcribed miRNAs obtained in the previous step.
PCR products produced according to these methods were further amplified by using T7 polymerase for in vitro transcription from the T7 promoter sequence in the 5′ forward amplification primer. This provided a “sense”-strand target for hybridization. In addition, this sense-strand reaction product contained a complementary sequence to the “capture sequence”.
The in vitro transcribed sense-strand miRNA-specific products were used as described in the next Example to interrogate a microarray comprising antisense miRNA probes in order to identify miRNA species expressed or overexpressed in NPC tumors.
The in vitro transcribed sense-strand miRNA-specific products prepared according to Example 1 were used to interrogate a microarray comprising antisense miRNA probes as follows.
Microarrays were prepared comprising probes that were antisense dimers of mature miRNA sequences taken from miRBase (microrna.sanger.ac.uk/), previously termed “the microRNA registry” (Griffiths-Jones, 2004, The microRNA Registry Nucl. Acids. Res. 32: Database Issue, D109-D111). Each miRNA probe sequence used in the microarray was modified at its 5′ end with a C6 amino linker that permitted the probe to be attached to aldehyde-coated slides for microarray fabrication. A total of two hundred seven probes from two hundred twenty-two human miRNAs and six probes for five EBV miRNAs (as present in the database as of April 2005) were spotted on a chip. Also spotted were seven probes from D. melanogaster miRNAs as controls (Table 1). Microarrays were printed in quadruplicate for each probe in an amount of 40 μM probe in 2.4×SSC on aldehyde-coated slides (ArrayIt SuperAldehyde Substrates, obtained from Telechem International, Inc., Sunnyvale, Calif., USA) using a BioRobotics MicroGrid II microarrayer (Genomic Solutions, Ann Arbor, Mich., USA). The microarrays were preprocessed according to the slide manufacturer's instructions.
Two hybridization steps were performed on these arrays: 1) sense target hybridization, and 2) capture sequence hybridization (illustrated in
After hybridization, the arrays were washed, spin-dried and the second hybridization was performed to detect the position in the array that had hybridized to an amplified miRNA species in the hybridization mixture. The washing condition used for both washes follows: (a) removed the LifterSlip by putting the array in a beaker containing 2×SSC, 0.2% SDS, where the solution being at 55° C. for the first hybridization and 42° C. for the second hybridization; (b) washed for 15 minutes in 2×SSC, 0.2% SDS; (c) then washed for 15 minutes in 2×SSC; (d) and then finally washed for 15 minutes in 0.5×SSC.
The second hybridization used a Cy3 3DNA molecule containing the “capture sequence” wherein these molecules contained an aggregate of approximately 900 fluorophores; these reagents and buffers were commercially available (34 μl vol containing 2.5 μl of 3DNA capture reagent, 14.5 μl water and 17 μl SDS-based hybridization buffer) (3DNA Array 900 Microarray detection kit, Genisphere Inc., Hatfield, Pa., USA). After the second hybridization at 42° C. for 4 hours, the arrays were again washed (conditions above), dried and scanned. Data was acquired with GenePix Pro 5.0 (Molecular Devices, Sunnyvale, Calif., USA). All hybridization buffers, wash conditions etc. used in the second detection reaction were provided by/according to Genisphere. The results of these assays, and further characterization of the miRNA species, are presented in Example 3.
Cellular and viral miRNAs in EBV-associated cancers such as NPC are candidate oncogenes that may contribute to the initiation or maintenance, or both, of tumors. Accordingly, the microarray methods described above were used to screen a large number of cellular and viral miRNAs for differential expression in NPC tumors. These assays were performed using microarrays prepared as described in Example 2, comprising two hundred twenty-two human miRNAs and for five viral miRNAs, which included all miRNAs identified as of April 2005. These assays were performed substantially as described above.
The results of these assays are given in Table 2. In these experiments, background-corrected, raw-scale expression intensity values were obtained via GenePix Pro 5.0 (Molecular Devices) after some manual adjustment to align and identify spots. Data from multiple microarrays were normalized using a version of quantile normalization (Bolstad et al., 2003, Bioinformatics 19:185-93) in which the expression value at the pth quantile on the ith microarray was replaced by the median of pth quantiles across the set of all 41 microarrays. Gene-specific hypothesis tests were applied to the quantile-normalized data in order to assess differential expression between tumor and normal microRNA profiles. To minimize false positive calls and retain robustness, multiple statistical tests (including Wilcoxon rank sum, t-test, raw scale, and t-test, log scale at 5% false discovery rate) were used to establish the significance of the differences in expression between tumor and normal tissue. In applying this statistical analysis, an miRNA species was determined to be differentially expressed if it was significantly different by all three tests, at the 5% false discovery rate. Gene-specific p-values were converted to q-values (Storey and Tibshirani, 2003, Proc Natl Acad Sci USA. 100:9440-5); the list containing genes with q-value <=5% is expected to have no more than 5% false positives.
For the miRNA results, robust differential expression was detected between tumor and normal tissues; in these analyses miRNAs expressed at very low levels, less than 800 relative fluorescence units (RFUs), in both tissue types were excluded from the analysis. Eight miRNAs showed a greater than five-fold differential in expression between normal and tumor tissues. Of these, six miRNAs (miR-29c, miR-34c, miR-34b, miR-212, miR-216 and miR-217) showed significantly higher expression in normal cells as compared to tumors and 2 (miR-151 and miR-192) showed significantly higher expression in tumors as compared to normal samples in this analysis (Table 3).
Hence stringent statistical criteria established eight human miRNAs to be differentially expressed between tumor and normal tissues.
The results shown in Example 3 identified eight human miRNAs that were significantly differentially expressed between normal and tumor tissues and that likely contribute to tumor phenotype. The assays described in this Example were performed to identify mRNA species whose expression is regulated by any of these eight miRNAs.
These assays were performed by applying two algorithms, both of which predicted targets by finding sequences in 3′ UTRs of mRNAs that match nucleotides 2 through 7 of the 5′ end of the identified miRNAs. The first, termed PicTar (Krek et al., 2005, Nat Genet. 37:495-500) also further refined its predictions by searching for target conservation in mammals (human, chimp, mouse, rat, dog) (pictar.bio.nyu.edu/cgi-bin/PicTar_vertebrate.cgi). The second algorithm, termed TargetScan (Lewis et al., (2003, Cell. 115:787-98), looked for conservation of target sites in vertebrates (www.targetscan.org). Targets predicted by both algorithms were considered in further analysis.
The target sites of miRNAs in mRNAs often are evolutionarily conserved and considering such conservation increases the reliability of identifying targets (Lewis et al., 2005, Cell. 120:15-20). Because these target sites are identified by a minimum perfect complementarity of only 7 to 8 nucleotides at the 5′ end of the miRNAs (the ‘seed’ sequence), these algorithms sometimes produce false-positive targets. In addition to regulating gene expression by inhibiting translation (which is thought to be the more common action of miRNAs), miRNAs can also regulate expression of a subset of their targets by decreasing mRNA stability (Yekta et al., Science. 304:594-596; Bagga et al., 2005, Cell. 122:553-563; and Wu et al., 2006, Proc Natl Acad Sci USA. 103:4034-4039). Such miRNA function should be evident in gene expression profiling data. Therefore, prior mRNA profiling (Sengupta et al., 2006, Cancer Res. 66:7999-8006) results were used to find bona fide targets among the large number of predicted target mRNAs of the eight highly differentially expressed miRNAs, by identifying those targets that accumulate differentially between tumor and normal samples.
None of the predicted target mRNAs for mir-151 and mir-192 showed differential mRNA accumulation. However, statistically significant differentially accumulating, candidate target mRNAs for the six miRNAs whose levels decreased in NPC were identified (Table 4). The largest set of differentially expressed predicted targets was associated with mir-29c. Mir-29c levels averaged one-fifth the level in NPC tumors as in normal nasopharyngeal epithelium (Table 3) and, correspondingly, the 15 differentially accumulating, predicted mir-29c target mRNAs accumulated to 2- to 6-fold higher levels in NPC tumors (Table 4). Strikingly, 10 of these 15 differentially accumulating candidate target mRNAs of mir-29c were involved in extracellular matrix synthesis or its functions, including 7 collagens, laminin γ1, fibrillin, and SPARC (secreted protein, acidic, cysteine-rich). Interestingly, two differentially expressed mir-29c targets, laminin γ1 and FUSIP1 (FUS interacting protein) mRNAs, also were predicted targets of mir-216 and mir-217, respectively, which like mir-29c were downregulated miRNAs in NPC tumors (Tables 3 and 4).
The seed sequence of mir-29c is identical to that of its two family members, mir-29a and mir-29b. These three mir-29 species vary in their last few 3′-end nucleotides. In addition, in close proximity to its seed sequence, mir-29a has a single nucleotide difference from mir-29b&c, giving mir-29c an overlapping but distinct list of predicted target mRNAs. Mir-29a is expressed at slightly higher levels than mir-29c in normal tissue, and its levels are moderately decreased in tumors. Mir-29b, predominantly targeted to the nucleus (Hwang et al., 2007, Science. 315:97-100), is expressed at one-fourth the level of mir-29c in normal nasopharyngeal epithelium. In NPC tumors, mir-29b and mir-29c have similar 4-fold to 5-fold decreased levels (Table 2). Thus, the levels of all three mir-29 family members are decreased in tumors, implying parallel effects on their shared targets.
The mechanism of action of miRNA-mediated gene expression regulation is understood to encompass not only modulating mRNA translation. This miRNA-mediated gene expression regulation may include, for example, decreasing mRNA translation or reducing stability of specific mRNAs (Yekta et al., 2004, Science. 304:594-6; Wu et al., 2006, Proc Natl Acad Sci USA. 103:4034-9). Thus, all predicted targets for these 8 miRNAs were cross checked for differential expression between NPC tumor samples and corresponding normal tissues (Sengupta et al., 2006, Cancer Res. 66: 7999-8006) to identify mRNAs that are downregulated in tissue (tumor/normal) where the miRNA is upregulated. Excluded from analysis were those mRNAs detected at low levels in both tumor and normal cells, to insure that only robust potential targets were considered. Target mRNAs for six of the eight miRNAs were found which showed downregulation in tissues where the miRNA was upregulated (Table 4). One miRNA, miR-29c had a group of target genes that were functionally related.
For many tumor cells, increased extracellular levels of collagens and/or laminins have been shown to induce increased invasiveness in culture and increased metastasis in animal models (Kaufman et al., 2005, Biophys J. 89:635-650; Koenig et al., 2006 Cancer Res. 66:4662-4671; Chintala et al., 1996, Cancer Lett 102:57-63; Kuratomi et al., 1999, Exp Cell Res. 249:386-395; Kuratomi et al., 2002, Br J Cancer. 86:1169-1173; Song et al., 1997, Int J Cancer. 71:436-441; Menke et al., 2001, Cancer Res. 61:3508-3517; Shintani et al., 2006, Cancer Res 66:11745-11753). Similarly, increased levels of collagens and laminins have been associated with an increased likelihood of clinical metastasis of multiple human solid tumors (Ramaswamy et al., 2003, Nat Genet 33:49-54). The results set forth herein, disclosing use of laser-capture to isolate tumor cells essentially free of stromal contaminants (Sengupta et al., 2006, Cancer Res. 66:7999-8006). indicated that NPC tumor cells upregulate mRNAs encoding collagens and laminins.
The capacity of the miRNA species miR-29c to regulate the target mRNAs identified above was confirmed as follows.
A precursor of miR-29c was introduced into human epithelial and liver cell lines Hela and HepG2 and the levels of the processed miRNA and its target mRNAs were assayed by quantitative real time PCR. The resulting changes in levels of the mature miRNA and its target mRNAs relative to their levels in untransfected cells were measured (Table 5). HeLa and HepG2 were transfected with miR-29c precursor molecules and negative controls (Ambion, Austin, Tex., USA) using TranslT-TKO reagent (Minis Bio Corporation, Madison, Wis., USA). Transfection efficiencies were monitored with LabelIT miRNA Labeling Kit (Minis Bio Corporation, Madison, Wis., USA). Levels of mature miR-29c in transfected and untransfected control cells were measured by stem-loop quantitative PCR (Chen et al., 2005, Nucleic Acids Res. 33:179) using TaqMan MicroRNA Assay and TaqMan MicroRNA Reverse Transcription Kits (Applied Biosystems, Foster City, Calif., USA). mRNA from untransfected cells and cells transfected with the negative control and miR-29c precursor were reverse transcribed using oligo-dT primers and SuperScript™ II Reverse Transcriptase (Invitrogen, Carlsbad, Calif., USA) and expression of miR-29c target genes was measured by quantitative real time PCR using QuantiTect SYBR Green PCR Kit (Qiagen, Valencia, Calif., USA). The primer sequences are listed in Table 6. All experimental manipulations disclosed in this Example were performed according to the manufacturers' instructions and as understood by one having skill in this art. All gene measurements were done 24 h post-transfection.
The transfected Hela and HepG2 cells had a 100- and 10-fold increase in their level of mature mirR-29c, respectively, as measured by stem loop quantitative real time PCR relative to untransfected cells or those transfected with a negative control precursor RNA that is processed into a randomized sequence not matching any known miRNA. In HeLa cells, 8 potential miR-29c target mRNAs were detected at high copy numbers. Another five (collagen 3A1, 4A1, 15A1, laminin γ1 and thymine-DNA glycosylase (TDG)) were reduced significantly by miR-29c transfection, as shown in
In addition, HepG2 cells showed significant, above-background measurements for additional miR-29c candidate targets collagen 1A2, fibrillin 1, SPARC and FUSIP1 mRNAs, revealing miR-29c-mediated reductions for all of those except SPARC (
To verify mir-29's regulation of target gene expression, 3′ UTRs containing mir-29c binding site sequence, were cloned into expression vectors containing a luciferase reporter gene. Specifically, 10 mir-29c target gene 3′ UTRs were cloned into a vector immediately downstream of a firefly luciferase gene. As a control, the GAPDH 3′UTR, which is not a mir-29c target, was cloned downstream of luciferase.
The firefly luciferase expression vector pGL2-control (Promega, Madison, Wis.) was modified by introducing silent mutations in a potential mir-29c binding sequence in the firefly luciferase ORF (nt positions 844-860) and by replacing the 3′UTR of the luciferase gene with a double stranded oligonucleotide linker to create a multiple cloning site (NotI-SpeI-PstI-BamHI-SalI) immediately downstream from the Firefly luciferase ORF, while removing the existing SalI site from the original plasmid. This new vector, pJBLuc3UTR (SEQ ID NO: 539), accommodated subsequent insertion of the entire 3′UTR sequences of 12 mRNAs: COL1A1 (SEQ ID NO: 540), COL1A2 (SEQ ID NO: 541), COL3A1 (SEQ ID NO: 542), COL4A1 (SEQ ID NO: 543), COL4A2 (SEQ ID NO: 544), COL15A1 (SEQ ID NO: 545), FUSIP1 isoform 1 (SEQ ID NO: 546) and 2 (SEQ ID NO: 547), GAPDH (SEQ ID NO: 548), LAMC1 (SEQ ID NO: 549), SPARC (SEQ ID NO: 550), and TDG (SEQ ID NO: 551). Full sequences are also provided for reference: COL1A1 (SEQ ID NO: 552), COL1A2 (SEQ ID NO: 553), COL3A1 (SEQ ID NO: 554), COL4A1 (SEQ ID NO: 555), COL4A2 (SEQ ID NO: 556), COL15A1 (SEQ ID NO: 557), FUSIP1 isoform 1 (SEQ ID NO: 558) and 2 (SEQ ID NO: 559), GAPDH (SEQ ID NO: 560), LAMC1 (SEQ ID NO: 561), SPARC (SEQ ID NO: 562), and TDG (SEQ ID NO: 563). (See Appendix 1 for the above-mentioned sequences). The 3′UTR sequences were PCR-amplified from oligo-d(T)-primed HeLa cDNA derived from 10 total RNA extracted using RNeasy reagents and protocol (Qiagen, Valencia, Calif.). cDNA was generated using the SuperScript™II cDNA synthesis kit (Invitrogen, Carlsbad, Calif.) according to instructions. PCRs contained a mixture of 0.25 U Vent DNA polymerase (New England Biolabs, Ipswich, Mass.) and 1.875 U Taq DNA polymerase (Promega, Madison, Wis.) in a 50 μl 1× Vent DNA polymerase buffer system supplemented with 1.5 mM MgCl2, 1 ng template plasmid, 100 μM of all four dNTPS and 25 pmoles of each of two primers. Upon 5 minutes denaturation at 95° C., 30 amplification cycles were used (1 min 95° C.-30 sec 55° C.-1 min/kbp 72° C.) followed by 10 min at 72° C. and refrigeration to 4° C. PCR-primers were designed to introduce SpeI or NheI-sites and SalI sites immediately upstream and downstream from the mRNA specific sequences, respectively, to facilitate subcloning between the SpeI and SalI sites of the modified luciferase expression vector using standard molecular biology procedures. Reporter plasmids for COL1A1, COL3A1, and COL4A2 3′UTRS then served as templates for PCR-mediated mutagenesis of all multiple mir-29c target sequences (
The reporter plasmids described above were transfected into HeLa cell using TransIT-HeLaMONSTER transfection reagents and conditions from Minis Bio Corporation (Madison, Wis.). 1.2×106 HeLa cells were co-transfected with 500 ng Firefly reporter plasmids and 250 ng internal reference Renilla luciferase reporter plasmid pRL-SV40 (Promega, Madison, Wis.) in a final transfection volume of 1050 μl. At 4 hours post plasmid transfection, culture medium was removed and cells were mock-transfected or transfected with 25 pmoles mir-29c precursor (Ambion, Austin, Tex.) using TransIT-TKO reagents under conditions recommended by the manufacturer (Minis Bio Corporation, Madison, Wis.) at a final transfection volume of 600 μl. Lysates were prepared at 24 hours post-transfection.
For dual luciferase reporter assays, transfected cells were lysed in 200 μl “passive lysis buffer” (Promega, Madison, Wis.) for 10 min at room temperature, scraped, resuspended, and cleared of nuclei and large cell debris by centrifugation at 10,000×g for 2 min at 4° C. Lysates were stored at −80° C. prior to analysis. 15 μl aliquots of the lysates were analyzed for Firefly luciferase activity and subsequently for Renilla luciferase activity using the Promega “Dual Luciferase Assay kit” for combined Firefly and Renilla luciferase assays as per accompanying instructions. Enzymatic activities were measured by luminometry using a Wallac 1420 Multilabel Counter (Victor3™V, Perkin Elmer, Waltham, Mass.). All measurements were normalized for Renilla luciferase activity to correct for variations in transfection efficiencies and non-mir-29c-specific effects of miRNA transfection on enzymatic activity.
For the experimental studies represented in
The magnitude of the mir-29c effects reported here for target mRNAs (
Nucleotide substitutions disrupting the mir-29c binding site(s) were introduced in the 3′ UTRs of collagen 1A1, 3A1, and 4A2 cloned downstream of the firefly luciferase gene (
In summary, miRNA expression profiling was performed in laser-microdissected NPC and normal surrounding epithelial cells using a sensitive assay specifically developed to detect miRNA expression from small samples limited in the amount of source tumor cells, the amount of miRNA or both. Eight of 207 assayed miRNAs displayed >5 fold differential expression levels in NPC cells compared to surrounding normal epithelium (Table 3). Using bioinformatic approaches candidate target genes of these 8 miRNAs were identified. Next, mRNA expression profiling was performed on these same specimens (Sengupta et al., 2006, Cancer Res. 66:7999-8006) further identifying candidate target genes that were differentially expressed, likely due to action of these miRNAs. Among the differentially expressed candidate target genes of the 8 miRNAs, those of mir-29c showed a group of 15 genes, 10 of which were extracellular matrix components involved in cell migration and metastasis (Table 4). In tumor cells, mir-29c levels were decreased >5 fold whereas these mRNAs were upregulated 2- to 6-fold.
Using multiple tissue culture-based assays (
All references cited herein are incorporated by reference. In addition, the invention is not intended to be limited to the disclosed embodiments of the invention. It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims.
This application is a continuation of U.S. Ser. No. 12/116,815, filed May 7, 2008, which claims priority to U.S. provisional application, Ser. No. 60/942,601, filed Jun. 7, 2007. Both applications are incorporated by reference herein in their entirety.
This invention was made with government support under CA097944 and CA022443 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 60942601 | Jun 2007 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12116815 | May 2008 | US |
| Child | 15013189 | US |
