The present technology relates to methods of diagnosing and treating human cancers, e.g., prostate cancer.
The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
There is considerable interest in understanding the function of RNA transcripts that do not code for proteins in eukaryotic cells. As evidenced by cDNA cloning projects and genomic tiling arrays, more than 90% of the human genome undergoes transcription but does not code for proteins. These transcriptional products are referred to as non-protein coding RNAs (ncRNAs). A variety of ncRNA transcripts, such as ribosomal RNAs, transfer RNAs, and spliceosomal RNAs, are essential for cell function. Similarly, a large number of short ncRNAs such as micro-RNAs (miRNAs), endogenous short interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs) and small nucleolar RNAs (snoRNAs) are also known to play important regulatory roles in eukaryotic cells. Recent studies have demonstrated a group of long ncRNA (lncRNA) transcripts that exhibit cell type-specific expression and localize into specific subcellular compartments. lncRNAs are also known to play an important roles during cellular development and differentiation supporting the view that they have been selected during the evolutionary process.
LncRNAs appear to have many different functions. In many cases, they seem to play a role in regulating the activity or localization of proteins, or serve as organizational frameworks for subcellular structures. In other cases, lncRNAs are processed to yield multiple small RNAs or they may modulate how other RNAs are processed.
Interestingly, lncRNAs can influence the expression of specific target proteins at specific genomic loci, modulate the activity of protein binding partners, direct chromatin-modifying complexes to their sites of action, and are post-transcriptionally processed to produce numerous 5′-capped small RNAs. Epigenetic pathways can also regulate the differential expression of lncRNAs. lncRNAs are misregulated in various diseases, including ischaemia, heart disease, Alzheimer's disease, psoriasis, and spinocerebellar ataxia type 8. This misregulation has also been shown in various types of cancers, such as breast cancer, colon cancer, prostate cancer, hepatocellular carcinoma and leukemia. One such lncRNA, DD3 (also known as PCA3), is listed as a specific prostate cancer biomarker. Recent studies have revealed the contribution of ncRNAs as proto-oncogenes, e.g. GAGE6, as tumor suppressor genes in tumorigenesis, and as drivers of metastatic transformation, e.g. HOTAIR in breast cancer.
Prostate cancer (PCa) is one of the leading causes of cancer deaths among American men. According to 2013 National Cancer Institute estimates, there will be 238,590 new prostate cancer diagnoses this year; for 29,720 men this is likely to be fatal. Although the incidence of prostate cancer has been steadily rising [2], with a concurrent increase in aggressive surgical management [3], most men have indolent disease for which conservative therapy or an active surveillance approach would be more appropriate and result in less treatment-related morbidity [1]. A contributing problem has been the widespread use of prostate specific antigen (PSA) testing, which has low specificity for cancer and cannot differentiate indolent and aggressive cancers; this has resulted in large numbers of unnecessary biopsies and overtreatment. There is therefore an urgent unmet need for a specific prognostic biomarker that can refine existing diagnostic methods.
The present technology is based on the discovery of the biomarkers for the early detection of prostate cancer to reduce over-treatment and accompanying morbidity.
In one aspect, the present technology provides for a method for accessing the progression of prostate cancer in a subject who is undergoing treatment for prostate cancer, which method comprises: (i) assessing the expression level of a long noncoding RNA in a biological sample obtained from the subject; (ii) comparing the expression level of the long noncoding RNA in the sample to a reference derived from the expression level of the long noncoding RNA in samples obtained from healthy subjects and determining the current condition of the subject; and (iii) for the subject determined to suffer from prostate cancer periodically repeating steps (i) and (ii) during treatment as a basis to determine the efficacy of said treatment by assessing whether the expression level of the long noncoding RNA in the subject is up-regulated or down-regulated, wherein a down-regulation in the expression level of the long noncoding RNA correlates to an improvement in the subject's condition.
In some embodiments, the long noncoding RNA is selected from the group consisting of SEQ ID NOs: 2-76. In some embodiments, the method further comprises assessing the expression level of SPRY4-IT1 (SEQ ID NO: 1).
In some embodiments, the expression level of the long noncoding RNA is assessed by evaluating the amount of the long noncoding RNA using a probe. In some embodiments, the biological sample comprises a tissue sample. In some embodiments, the tissue sample is a prostatic adenocarcinoma tissue sample. In some embodiments, the prostate cancer is early stage prostate cancer.
In some embodiments, the long noncoding RNA is XLOC_007697 (SEQ ID NO: 2). In some embodiments, the long noncoding RNA is XLOC_009911 (SEQ ID NO: 3). In some embodiments, the long noncoding RNA is XLOC_008559 (SEQ ID NO: 4). In some embodiments, the long noncoding RNA is XLOC_005327 (SEQ ID NO: 5). In some embodiments, the long noncoding RNA is LOC100287482 (SEQ ID NO: 6).
In another aspect, the present technology provides for a method for treating prostate cancer in a patient diagnosed as having prostate cancer comprising administering to the patient an effective amount of a therapeutic agent that reduces or down-regulates the expression level of a long noncoding RNA.
In some embodiments, the long noncoding RNA is selected from the group consisting of SEQ ID NOs: 2-76. In some embodiments, the long noncoding RNA expression is reduced or down-regulated in prostate cancer cells. In some embodiments, the long noncoding RNA expression is reduced by at least about 50%, 60%, 70%, 80% or 90%. In some embodiments, the therapeutic agent is an siRNA. In some embodiments, the therapeutic agent is contained within a liposome.
In yet another aspect, the present technology provides for a method for determining a treatment regimen for a patient with prostate cancer which method comprises: identifying whether said cancer is aggressive or indolent by identifying one or more of markers for aggressive prostate cancer said marker is one or more of PSA isoforms, kallikreins, GSTP1, AMACR, ERG, gene fusions involving ETS-related genes, PCA3, or a combination thereof; treating said cancer with a regimen consistent with whether the cancer is aggressive or indolent.
In some embodiments, the progress of said treatment regimen is monitored by further evaluating the presence and quantity of one or more of said markers in said patient and optionally adjusting the treatment protocol based on said evaluation.
In some embodiments, the treatment regimen is one or more of open prostatectomy, minimally invasive laparoscopic robotic surgery, intensity modulated radiation therapy (IMRT), proton therapy, brachytherapy, cryotherapy, molecular-targeted therapy, vaccine therapy and gene therapy, hormone therapy, active surveillance, or a combination thereof.
In yet another aspect, the present technology provides for a method for detecting prostate cancer in a patient suspected of having prostate cancer, which method comprises: (i) assessing the expression level of a long noncoding RNA in a biological sample obtained from said patient; (ii) comparing the expression level of the long noncoding RNA in the sample to a reference derived from the expression level of the long noncoding RNA in samples obtained from healthy subjects; (iii) identifying said patient as having prostate cancer when the expression level of the long noncoding RNA in said patient is greater than the reference or identifying said patient as not having prostate cancer when the expression level of the long noncoding RNA is equal or less than the reference.
In some embodiments, the patient is suspected of prostate cancer based on the patient's prostate specific antigen (PSA) Score, the Myriad Prolaris Assay (MPA) Score, the Oncotype DX Genomic Prostate Score (GPS), or the Cancer of the Prostate Risk Assessment (CAPRA) Score.
In yet another aspect, the present technology provides for a method for differentiating indolent and aggressive prostate cancer, which method comprises: identifying the aggressive prostate cancer based on the expression of one or more of aggressive tumor-predictive genes associated with the aggressive prostate cancer; and identifying the indolent prostate cancer based on the lack of the expression or the low expression of one or more of aggressive tumor-predictive genes associated, and wherein the expression of aggressive tumor-predictive genes is determined by one or more of prostate specific antigen (PSA) Score, the Myriad Prolaris Assay (MPA) Score, the Oncotype DX Genomic Prostate Score (GPS), the Cancer of the Prostate Risk Assessment (CAPRA) Score, or a combination thereof.
In yet another aspect, the present technology provides for a kit comprising a composition comprising a long noncoding RNA, and instructions for use, wherein the long noncoding RNA is selected from the group consisting of SEQ ID NOs: 2-76.
The present invention relates generally to identifying and characterizing long non-coding RNAs (“lncRNAs”) that are differentially expressed in cancer cells, particularly in prostate cancer, as compared to normal tissue. The identification of cancer-associated lncRNAs and the investigation of their molecular and biological functions aids in understanding the molecular etiology of cancer and its progression.
As used herein, the term “nucleic acid molecule” or “nucleic acid” refer to an oligonucleotide, nucleotide or polynucleotide. A nucleic acid molecule may include deoxyribonucleotides, ribonucleotides, modified nucleotides or nucleotide analogs in any combination.
As used herein, the term “nucleotide” refers to a chemical moiety having a sugar (modified, unmodified, or an analog thereof), a nucleotide base (modified, unmodified, or an analog thereof), and a phosphate group (modified, unmodified, or an analog thereof). Nucleotides include deoxyribonucleotides, ribonucleotides, and modified nucleotide analogs including, for example, locked nucleic acids (“LNAs”), peptide nucleic acids (“PNAs”), L-nucleotides, ethylene-bridged nucleic acids (“ENAs”), arabinoside, and nucleotide analogs (including abasic nucleotides).
As used herein, the term “short interfering nucleic acid” or “siNA” refers to any nucleic acid molecule capable of down regulating (i.e., inhibiting) gene expression in a mammalian cells (preferably a human cell). siNA includes without limitation nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA).
As used herein, the term “sense region” refers to a nucleotide sequence of a siNA molecule complementary (partially or fully) to an antisense region of the siNA molecule. Optionally, the sense strand of a siNA molecule may also include additional nucleotides not complementary to the antisense region of the siNA molecule.
As used herein, the term “ectopic expression” refers to the occurrence of gene expression or the occurrence of a level of gene expression in a tissue in which it is not generally expressed, or not generally expressed at such a level.
As used herein, the term “antisense region” refers to a nucleotide sequence of a siNA molecule complementary (partially or fully) to a target nucleic acid sequence. Optionally, the antisense strand of a siNA molecule may include additional nucleotides not complementary to the sense region of the siNA molecule.
As used herein, the term “duplex region” refers to the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 bases on each strand are complementary or substantially complementary, such that the “duplex region” consists of 19 base pairs. The remaining base pairs may, for example, exist as 5′ and/or 3′ overhangs.
An “abasic nucleotide” conforms to the general requirements of a nucleotide in that it contains a ribose or deoxyribose sugar and a phosphate but, unlike a normal nucleotide, it lacks a base (i.e., lacks an adenine, guanine, thymine, cytosine, or uracil). Abasic deoxyribose moieties include, for example, abasic deoxyribose-3′-phosphate; 1,2-dideoxy-D-ribofuranose-3-phosphate; 1,4-anhydro-2-deoxy-D-ribitol-3-phosphate.
As used herein, the term “inhibit”, “down-regulate”, or “reduce,” with respect to gene expression, means that the level of RNA molecules encoding one or more proteins or protein subunits (e.g., mRNA) is reduced below that observed in the absence of the inhibitor. Expression may be reduced by at least 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or below the expression level observed in the absence of the inhibitor.
A group of differentially expressed long noncoding RNAs (IncRNAs) are identified in prostate cancer cell lines and patient samples using DNA microarrays, and performed confirmatory analysis using qRT-PCR and RNA-FISH. Several highly upregulated IncRNAs were further tested in prostatic adenocarcinoma tissue samples (Gleason score >6.0) and compared to matched normal tissues. AK024556, XLOC-007697, LOC100506411, LOC100287482, XLOC-001699, XLOC-005327, XLOC-008659, and XLOC-009911 were confirmed as significantly upregulated in patient samples,
In some embodiments, the IncRNA that is significantly upregulated in prostate cancer cells comparing to a reference level determined in a healthy subject is one or more of SEQ ID NOs: 1-76, or a combination thereof. In some embodiments, the IncRNA that is significantly upregulated in prostate cancer cells is XLOC_007697 (SEQ ID NO: 2). In some embodiments, the IncRNA that is significantly upregulated in prostate cancer cells is XLOC_009911 (SEQ ID NO: 3). In some embodiments, the IncRNA that is significantly upregulated in prostate cancer cells is XLOC_008559 (SEQ ID NO: 4). In some embodiments, the IncRNA that is significantly upregulated in prostate cancer cells is XLOC_005327 (SEQ ID NO: 5). In some embodiments, the IncRNA that is significantly upregulated in prostate cancer cells is LOC100287482 (SEQ ID NO: 6).
AK024556, also known as SPRY4-IT1, is an intronic IncRNA originating from the first intron of the SPRY4 gene) was previously reported to be upregulated in primary human melanomas and cell lines. SPRY4-IT1 was not expressed in LNCaP cells due to the epigenetic modification of the SPRY4 promoter by CpG island methylation. Furthermore, epigenetic silencing was reversed by treatment with 5-aza-2′-deoxycytidine (a DNA methyltransferase inhibitor) and resulted in upregulation of SPRY4 and SPRY4-IT1, indicating that SPRY4 and SPRY4-IT1 are epigenetically co-regulated. siRNA knockdown of SPRY4-IT1 inhibited proliferation and invasion, and increased apoptosis, in PC3 cells. Chromogenic in situ hybridization (CISH) assay was developed to detect SPRY4-IT1 in patient samples. The present technology is useful for prostate cancer diagnosis in a clinical setting. Results are reported here to support the notion that IncRNAs are potential diagnostic biomarkers for prostate cancers with have a role in prostate carcinogenesis.
To address the need for a specific prognostic biomarker that can refine existing diagnostic methods, several diagnostic and predictive biomarkers are being actively investigated or are in clinical use [4], including the use of PSA isoforms, kallikreins, and measurement of the expression of genes that are associated with prostate cancer (such as GSTP1, AMACR, ERG, and gene fusions involving ETS-related genes). In particular, PCA3, a long non-coding RNA (IncRNA), has shown promise for the urinary detection of prostate cancer with superior specificity to PSA [42].
LncRNAs are RNA transcripts >200 nucleotides in length [5, 6], many of which exhibit cell type-specific expression [7-9] and are localized to specific subcellular compartments [10-14]. A number of IncRNAs are known to play important roles during cellular development and differentiation [15-17], supporting the view that they are under evolutionary selection [18-21].
LncRNAs can influence the expression of target proteins at specific genomic loci [22-25], modulate the activity of protein binding partners [26-28], direct chromatin-modifying complexes to their sites of action, and undergo post-transcriptional processing to produce numerous 5′-capped small RNAs [10, 29]. Like microRNAs (miRNAs), IncRNAs are dysregulated in various diseases, including ischemia, heart disease [30, 31], Alzheimer's disease [32], psoriasis [33], spinocerebellar ataxia type 8 [34, 35], and several cancers such as breast cancer [16, 36, 37], colon cancer [38], prostate cancer [39], hepatocellular carcinoma [40, 41], and leukemia [40].
SPRY4-IT1 is upregulated in human melanomas, and siRNA-mediated knockdown of SPRY4-IT1 in melanoma cells alters cellular growth and differentiation and increases the rate of apoptosis [43]. The differential expression of several prostate cancer specific IncRNAs and their expression are investigated in prostate cancer cell lines, normal epithelial cells, and prostate cancer patient samples matched with normal tissues, and explore the molecular function of the IncRNA SPRY4-IT1 in prostate cancer cells using siRNA knockdown and cellular assays.
In some embodiments, the reduction or inhibition or down-regulation of one or more of the IncRNAs (i.e., SEQ ID NOs: 1-76, or a combination thereof) that are significantly upregulated in prostate cancer cells influence the expression of target proteins at specific genomic loci. In some embodiments, the reduction or inhibition or down-regulation of one or more of the IncRNAs (i.e., SEQ ID NOs: 1-76, or a combination thereof) that are significantly upregulated in prostate cancer cells modulate the activity of protein binding partners. In some embodiments, the reduction or inhibition or down-regulation of one or more of the IncRNAs (i.e., SEQ ID NOs: 1-76, or a combination thereof) that are significantly upregulated in prostate cancer cells direct chromatin-modifying complexes to their sites of action. In some embodiments, the reduction or inhibition or down-regulation of one or more of the IncRNAs (i.e., SEQ ID NOs: 1-76, or a combination thereof) that are significantly upregulated in prostate cancer cells undergo post-transcriptional processing to produce 5′-capped small RNAs. In some embodiments, the IncRNA is XLOC_007697 (SEQ ID NO: 2). In some embodiments, the IncRNA is XLOC_009911 (SEQ ID NO: 3). In some embodiments, the IncRNA is XLOC_008559 (SEQ ID NO: 4). In some embodiments, the IncRNA is XLOC_005327 (SEQ ID NO: 5). In some embodiments, the IncRNA is LOC100287482 (SEQ ID NO: 6).
RNA Interference and siNA
RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Post-transcriptional gene silencing is believed to be an evolutionarily-conserved cellular mechanism for preventing expression of foreign genes that may be introduced into the host cell (Fire et al., 1999, Trends Genet., 15, 358). Post-transcriptional gene silencing may be an evolutionary response to the production of double-stranded RNAs (dsRNAs) resulting from viral infection or from the random integration of transposable elements (transposons) into a host genome. The presence of dsRNA in cells triggers the RNAi response that appears to be different from other known mechanisms involving double stranded RNA-specific ribonucleases, such as the interferon response that results from dsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylate synthetase resulting in non-specific cleavage of mRNA by ribonuclease L (see for example U.S. Pat. No. 6,107,094; 5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17, 503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).
The presence of long dsRNAs in cells stimulates the activity of dicer, a ribonuclease III enzyme (Bass, 2000, Cell, 101, 235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000, Nature, 404, 293). Dicer processes long dsRNA into double-stranded short interfering RNAs (siRNAs) which are typically about 21 to about 23 nucleotides in length and include about 19 base pair duplexes (Zamore et al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Elbashir et al., 2001, Genes Dev., 15, 188).
Single-stranded RNA, including the sense strand of siRNA, trigger an RNAi response mediated by an endonuclease complex known as an RNA-induced silencing complex (RISC). RISC mediates cleavage of this single-stranded RNA in the middle of the siRNA duplex region (i.e., the region complementary to the antisense strand of the siRNA duplex) (Elbashir et al., 2001, Genes Dev., 15, 188).
In certain embodiments, the siNAs may be a substrate for the cytoplasmic Dicer enzyme (i.e., a “Dicer substrate”) which is characterized as a double stranded nucleic acid capable of being processed in vivo by Dicer to produce an active nucleic acid molecules. The activity of Dicer and requirements for Dicer substrates are described, for example, U.S. 2005/0244858. Briefly, it has been found that dsRNA, having about 25 to about 30 nucleotides, effective result in a down-regulation of gene expression. Without wishing to be bound by any theory, it is believed that Dicer cleaves the longer double stranded nucleic acid into shorter segments and facilitates the incorporation of the single-stranded cleavage products into the RNA-induced silencing complex (RISC complex). The active RISC complex, containing a single-stranded nucleic acid cleaves the cytoplasmic RNA having complementary sequences.
It is believed that Dicer substrates must conform to certain general requirements in order to be processed by Dicer. The Dicer substrates must of a sufficient length (about 25 to about 30 nucleotides) to produce an active nucleic acid molecule and may further include one or more of the following properties: (i) the dsRNA is asymmetric, e.g., has a 3′ overhang on the first strand (antisense strand) and (ii) the dsRNA has a modified 3′ end on the antisense strand (sense strand) to direct orientation of Dicer binding and processing of the dsRNA to an active siRNA. The Dicer substrates may be symmetric or asymmetric. For example, Dicer substrates may have a sense strand includes 22-28 nucleotides and the antisense strand may include 24-30 nucleotides, resulting in duplex regions of about 25 to about 30 nucleotides, optionally having 3′-overhangs of 1-3 nucleotides.
Dicer substrates may have any modifications to the nucleotide base, sugar or phosphate backbone as known in the art and/or as described herein for other nucleic acid molecules (such as siNA molecules).
The RNAi pathway may be induced in mammalian and other cells by the introduction of synthetic siRNAs that are 21 nucleotides in length (Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., WO 01/75164; incorporated by reference in their entirety). Other examples of the requirements necessary to induce the down-regulation of gene expression by RNAi are described in Zamore et al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429; Kreutzer et al., WO 00/44895; Zernicka-Goetz et al., WO 01/36646; Fire, WO 99/32619; Plaetinck et al., WO 00/01846; Mello and Fire, WO 01/29058; Deschamps-Depaillette, WO 99/07409; and Li et al., WO 00/44914; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831; each of which is hereby incorporated by reference in its entirety.
Briefly, an siNA nucleic acid molecule can be assembled from two separate polynucleotide strands (a sense strand and an antisense strand) that are at least partially complementary and capable of forming stable duplexes. The length of the duplex region may vary from about 15 to about 49 nucleotides (e.g., about 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides). Typically, the antisense strand includes nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule. The sense strand includes nucleotide sequence corresponding to the target nucleic acid sequence which is therefore at least substantially complementary to the antisense stand. Optionally, an siNA is “RISC length” and/or may be a substrate for the Dicer enzyme. Optionally, an siNA nucleic acid molecule may be assembled from a single polynucleotide, where the sense and antisense regions of the nucleic acid molecules are linked such that the antisense region and sense region fold to form a duplex region (i.e., forming a hairpin structure).
siNAs may be blunt-ended on both sides, have overhangs on both sides or a combination of blunt and overhang ends. Overhangs may occur on either the 5′- or 3′-end of the sense or antisense strand. Overhangs typically consist of 1-8 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides each) and are not necessarily the same length on the 5′- and 3′-end of the siNA duplex. The nucleotide(s) forming the overhang need not be of the same character as those of the duplex region and may include deoxyribonucleotide(s), ribonucleotide(s), natural and non-natural nucleobases or any nucleotide modified in the sugar, base or phosphate group such as disclosed herein.
The 5′- and/or 3′-end of one or both strands of the nucleic acid may include a free hydroxyl group or may contain a chemical modification to improve stability. Examples of end modifications (e.g., terminal caps) include, but are not limited to, abasic, deoxy abasic, inverted (deoxy) abasic, glyceryl, dinucleotide, acyclic nucleotide, amino, fluoro, chloro, bromo, CN, CF, methoxy, imidazole, carboxylate, thioate, C1 to C10 lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF3, OCN, O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2, N3; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino or substituted silyl, as, among others, described in European patents EP 586,520 and EP 618,925.
siNA molecules optionally may contain one or more chemical modifications to one or more nucleotides. There is no requirement that chemical modifications are of the same type or in the same location on each of the siNA strands. Thus, each of the sense and antisense strands of an siNA may contain a mixture of modified and unmodified nucleotides. Modifications may be made for any suitable purpose including, for example, to increase RNAi activity, increase the in vivo stability of the molecules (e.g., when present in the blood), and/or to increase bioavailability.
Suitable modifications include, for example, internucleotide or internucleoside linkages, dideoxyribonucleotides, 2′-sugar modification including amino, fluoro, methoxy, alkoxy and alkyl modifications; 2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base” nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, biotin group, and terminal glyceryl and/or inverted deoxy abasic residue incorporation, sterically hindered molecules, such as fluorescent molecules and the like. Other nucleotides modifiers could include 3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC), 2′,3′-didehydro-2′,3′-dideoxythymidi-ne (d4T) and the monophosphate nucleotides of 3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxy-3′-thiacytidine (3TC) and 2′,3′-didehydro-2′,3′-dide-oxythymidine (d4T).
Other suitable modifications include, for example, locked nucleic acid (LNA) nucleotides (e.g., 2′-0, 4′-C-methylene-(D-ribofuranosyl) nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azido nucleotides, and 2′-O-methyl nucleotides (WO 00/47599, WO 99/14226, WO 98/39352, and WO 2004/083430).
Chemical modifications also include terminal modifications on the 5′ and/or 3′ part of the oligonucleotides and are also known as capping moieties. Such terminal modifications are selected from a nucleotide, a modified nucleotide, a lipid, a peptide, and a sugar.
Chemical modifications also include L-nucleotides. Optionally, the L-nucleotides may further include at least one sugar or base modification and/or a backbone modification as described herein.
Nucleic acid molecules disclosed herein (including siNAs and Dicer substrates) may be administered with a carrier or diluent or with a delivery vehicle which facilitate entry to the cell. Suitable delivery vehicles include, for example, viral vectors, viral particles, liposome formulations, and lipofectin.
Methods for the delivery of nucleic acid molecules are described in Akhtar et al., Trends Cell Bio., 2: 139 (1992); Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, (1995), Maurer et al., Mol. Membr. Biol., 16: 129-140 (1999); Hofland and Huang, Handb. Exp. Pharmacol., 137: 165-192 (1999); and Lee et al., ACS Symp. Ser., 752: 184-192 (2000); U.S. Pat. Nos. 6,395,713; 6,235,310; 5,225,182; 5,169,383; 5,167,616; 4,959217; 4.925,678; 4,487,603; and 4,486,194; WO 94/02595; WO 00/03683; WO 02/08754; and U.S. 2003/077829.
Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins (see e.g., Gonzalez et al., Bioconjugate Chem., 10: 1068-1074 (1999); WO 03/47518; and WO 03/46185), poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see for example U.S. Pat. No. 6,447,796 and U.S. 2002/130430), biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (WO 00/53722). Alternatively, the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump. Direct injection of the nucleic acid molecules of the invention, whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., Clin. Cancer Res., 5: 2330-2337 (1999) and WO 99/31262. The molecules of the instant invention can be used as pharmaceutical agents.
Nucleic acid molecules may be complexed with cationic lipids, packaged within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complexes can be locally administered to relevant tissues ex vivo, or in vivo through direct dermal application, transdermal application, or injection, with or without their incorporation in biopolymers. Delivery systems include surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes).
Nucleic acid molecules may be formulated or complexed with polyethylenimine (e.g., linear or branched PEI) and/or polyethylenimine derivatives, including for example polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives, grafted PEIs such as galactose PEI, cholesterol PEI, antibody derivatized PEI, and polyethylene glycol PEI (PEG-PEI) derivatives thereof (see, for example Ogris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003, Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, Pharmaceutical Research, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22, 46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Peterson et al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999, Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNAS USA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release, 60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274, 19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; U.S. Pat. No. 6,586,524 and U.S. 2003/0077829).
Delivery systems may include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer. Examples of liposomes which can be used in this invention include the following: (1) CellFectin, 1:1.5 (M/M) liposome formulation of the cationic lipid N,NI,NII,NIII-tetramethyl-N,NI,NII,NIII-tetrapalmit-y-spermine and dioleoyl phosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2:1 (M/M) liposome formulation of a cationic lipid and DOPE (Glen Research); (3) DOTAP (N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate) (Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposome formulation of the polycationic lipid DOSPA, the neutral lipid DOPE (GIBCO BRL) and Di-Alkylated Amino Acid (DiLA2).
Therapeutic nucleic acid molecules may be expressed from transcription units inserted into DNA or RNA vectors. Recombinant vectors can be DNA plasmids or viral vectors. Nucleic acid molecule expressing viral vectors can be constructed based on, but not limited to, adeno-associated virus, retrovirus, adenovirus, or alphavirus. The recombinant vectors are capable of expressing the nucleic acid molecules either permanently or transiently in target cells. Delivery of nucleic acid molecule expressing vectors can be systemic, such as by intravenous, subcutaneous, or intramuscular administration.
Expression vectors may include a nucleic acid sequence encoding at least one nucleic acid molecule disclosed herein, in a manner which allows expression of the nucleic acid molecule. For example, the vector may contain sequence(s) encoding both strands of a nucleic acid molecule that include a duplex. The vector can also contain sequence(s) encoding a single nucleic acid molecule that is self-complementary and thus forms a nucleic acid molecule. Non-limiting examples of such expression vectors are described in Paul et al., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina et al., 2002, Nature Medicine. An expression vector may encode one or both strands of a nucleic acid duplex, or a single self-complementary strand that self hybridizes into a nucleic acid duplex. The nucleic acid sequences encoding nucleic acid molecules can be operably linked to a transcriptional regulatory element that results expression of the nucleic acid molecule in the target cell. Transcriptional regulatory elements may include one or more transcription initiation regions (e.g., eukaryotic pol I, II or III initiation region) and/or transcription termination regions (e.g., eukaryotic pol I, II or III termination region). The vector can optionally include an open reading frame (ORF) for a protein operably linked on the 5′ side or the 3′-side of the sequence encoding the nucleic acid molecule; and/or an intron (intervening sequences).
The nucleic acid molecules or the vector construct can be introduced into the cell using suitable formulations. One preferable formulation is with a lipid formulation such as in Lipofectamine™ 2000 (Invitrogen, CA, USA), vitamin A coupled liposomes (Sato et al. Nat Biotechnol 2008; 26:431-442, PCT Patent Publication No. WO 2006/068232). Lipid formulations can also be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as are known in the art. When the formulation is suitable for administration into animals such as mammals and more specifically humans, the formulation is also pharmaceutically acceptable. Pharmaceutically acceptable formulations for administering oligonucleotides are known and can be used. In some instances, it may be preferable to formulate dsRNA in a buffer or saline solution and directly inject the formulated dsRNA into cells, as in studies with oocytes. The direct injection of dsRNA duplexes may also be done. Suitable methods of introducing dsRNA are provided, for example, in U.S. 2004/0203145 and U.S. 20070265220.
Polymeric nanocapsules or microcapsules facilitate transport and release of the encapsulated or bound dsRNA into the cell. They include polymeric and monomeric materials, especially including polybutylcyanoacrylate. The polymeric materials which are formed from monomeric and/or oligomeric precursors in the polymerization/nanoparticle generation step, are per se known from the prior art, as are the molecular weights and molecular weight distribution of the polymeric material which a person skilled in the field of manufacturing nanoparticles may suitably select in accordance with the usual skill.
Nucleic acid moles may be formulated as a microemulsion. A microemulsion is a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution. Typically microemulsions are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a 4th component, generally an intermediate chain-length alcohol to form a transparent system. Surfactants that may be used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules.
The present methods, thus generally described, will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present methods and kits.
To identify which IncRNAs are differentially expressed in prostate cancer compared to normal prostatic epithelium, total RNA from human prostate epithelial cells and the prostate cancer cell line PC3 were screened using Ncode human microarrays. The Ncode human ncRNA microarray is designed to interrogate 12,784 IncRNAs and the expression of 25,409 mRNA target protein-coding genes. In addition, genome-wide expression analysis was performed on total RNA extracted from two prostate cancer cell lines (PC3 and LNCaP) and epithelial cells using the Agilent SurePrint G3 Human Gene Expression v2 microarray. This array measures expression of 16,472 IncRNAs and 34,127 mRNAs genes, and has an overlap of 460 lncRNAs and 8,877 mRNAs with the Ncode array. Therefore, by using these two arrays, a total of 28,796 IncRNAs and 50,659 mRNAs were examined.
AS shown in
Interestingly, SPRY4-IT1 was previously identified as one of the highly upregulated IncRNAs in human melanoma cells [43]. qRT-PCR analysis further confirmed that SPRY4-IT1 was upregulated over 100-fold in PC3 cells compared to prostatic epithelial cells (
An examination of the SPRY4 gene reveals that only one CpG island exists within its genomic locus. This island is present ˜900 bp upstream of the transcriptional start site (TSS; containing 11 CpG sequences in a 139 bp region;
After treatment of LNCaP cells with 5-aza-2′-deoxycytidine, half of the cell samples were bisulphite sequenced. The majority of methylation at this CpG island was depleted (89% methylation vs 30% after treatment;
Since the global IncRNA expression profile of prostate cancer has not been fully established, IncRNA expression profiles in prostate tissue samples from patients with prostate cancer were investigated. Ten paired (tumor and adjacent normal tissue) frozen biopsy specimens were obtained and total RNA profiled using the Agilent SurePrint G3 Human Gene Expression v2 microarray. Hierarchical clustering of the differentially expressed genes is shown in
SPRY4-IT1 expression levels were measured by qRT-PCR in a total of 18 matched normal prostate and prostatic adenocarcinoma tissue samples, with expression values normalized to 1 in the matched normal tissue. The expression of SPRY4-IT1 was variable in both normal and cancer tissues, probably due to variability in tissue composition (i.e. epithelial and stromal composition) and variable expression per cell. However, SPRY4-IT1 was significantly upregulated in cancerous tissue (
Since SPRY4-IT1 and SPRY4 can both be regulated by methylation of the same promoter (
Having confirmed that SPRY4-IT1 is overexpressed in primary prostatic adenocarcinoma by both ddPCR and qRT-PCR, SPRY4-IT1 expression in situ was visualized using RNA-CISH of tissue sections. Two matched tissue samples were selected for RNA-CISH and simultaneous comparison by qRT-PCR. There was a large difference in expression (an average increase of ˜7-fold) between the tumors and matched normal tissues (
RNA-CISH was performed on a prostate cancer tissue array in order to confirm specificity of expression in prostatic adenocarcinoma and assess associations with Gleason grading. SPRY4-IT1 expression was easily detected in all adenocarcinoma samples (Gleason scores 6 (3+3), 7 (3+4), 8 (4+4), 9 (5+4 & 4+5), & 10 (5+5)). However there was little or no staining in either normal (no cancer in the patient) or normal tissue adjacent to the cancer. These data indicate that SPRY4-IT1 expression is specific to adenocarcinoma and can be detected using standard clinical staining procedures, suggesting that this biomarker may be a viable diagnostic tool.
Previous study of SPRY4-IT1 in melanoma indicated that loss resulted in several negative phenotypes in the SPRY4-IT1-expressing cell lines examined [43].
To establish whether knockdown had similar effects in prostate cancer cells, PC3 cells were transfected with siRNAs specific to SPRY4-IT1. qRT-PCR indicated that knockdown equal to ˜40% loss of SPRY4-IT1 was achieved after 48 hours at both 100 nM and 200 nM siRNA concentrations (
All experiments described in this manuscript utilized at least one of the following human cell lines: prostate epithelial cells (ScienCell, HPrEpiC, Cat No 4410), PPC1, 22Rv1 (ATCC® CRL-2505™), DU-145 (ATCC® HTB-81™), LNCaP (ATCC® CRL1740™) and PC3 (ATCC® CRL-7934™) prostate cancer cell lines.
Prostate epithelial cells were grown in Prostate Epithelial Cell Medium (ScienCell, PEpiCM, Cat No 4411), whereas the prostate cancer cell lines were grown in DMEM (Invitrogen, Carlsbad, Calif.), supplemented with 10% FBS and Penicillin/Streptomycin.
The purity and integrity of the total RNA were analyzed on RNA Nano chip (Agilent Technologies) using Eukaryote Total RNA Nano series protocol. The total RNA was subjected to single round of linear IVT-amplification and labeled with Cy3-labeled CTP using One-Color Low Input Quick Amp Labeling Kit (Ambion). The resulting Cy3 dye incorporated antisence RNA (aRNA) was quantified using ND-1000 spectrophotometer (Nano Drop Technologies) and 600 ng of labeled aRNA was hybridized onto Ncode human ncRNA microarray (Life Technologies) or Agilent SurePrint G3 Human Gene Expression v2 (Agilent Technologies). After hybridization, the arrays were washed following the manufacturer's protocol using Gene Expression Wash Pack (Agilent Technologies) and scanned using the Agilent C Scanner. The intensities of the scanned fluorescence images were extracted with Agilent Feature Extrcation software version 10.7.3.1.
Total RNA from all cell lines was isolated using the Trizol method (Invitrogen/Life Technologies) with all quantification and integrity analysis performed with the NanoDropND-100 spectrometer (Thermo scientific, Wilminton, Del., USA). RNA (2 ug) was then used for cDNA synthesis in a 20 uL reaction volume using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster city, CA, USA). For detection of SPRY4-IT1 and SPRY4, qRT-PCR was performed in triplicate using a Power SYBR Green PCR master mix (Applied Biosystems, Warrington, UK) in the 7500 Real-Time PCR system (Applied Biosystems, Foster city, CA, USA). A final reaction volume of 20 ul was used, containing 2 ul of cDNA template, 10 ul of 2× Power SYBR Green PCR master mix, and 0.2 uM of each primer. The reaction was subjected to denaturation at 95° C. for 10 min followed by 40 cycles of denaturation at 95° C. for 15 sec and annealing at 58° C. for 1 min. SDS1.2.3 software (Applied Biosystems, Foster city, CA, USA) was used for comparative Ct analysis with GAPDH serving as the endogenous control.
Locked nucleic acid (LNA) modified probes for human IncRNA SPRY4-IT1 (TCCACTGGGCATATTCTAAAA), SPRY4 (GATGTTGCAACCACTGCCTGG) and a negative/scramble control (GTGTAACACGTCTATACGCCCA, miRCURY-LNA detection probe, Exiqon) containing biotin labels were used for RNA-FISH (Khaitan et al, 2011). In situ hybridization was then performed using the RiboMap ISH kit (Ventana Medical Systems, Inc.) using a Ventana machine. Cells in suspension were diluted to 10,000 cells/100 uL, pipetted on to autoclaved glass slides and allowed to adhere for 4 hours. The slides were then submerged in cell media (as above methods), then the following day removed from the media, washed with PBS and fixed in 4% paraformaldehyde/5% acetic acid. The slides were then subjected to the hydrochloride-based RiboClear reagent (Ventana Medical Systems) for 10′ at 37° C., followed by the ready-to-use protease 3 reagent. Cells were hybridized with antisense LNAriboprobe (40 nmol/L) using RiboHybe hybridization buffer (Ventana Medical Systems) for 2 hours at 58° C. after the initial denaturing prehybridization step for 4′ at 80° C. The slides were then treated to a low-stringency wash with 0.1% SSC (Ventana Medical Systems) for 4′ at 60° C. and 2 additional wash steps with 1% SSC for 4′ at 60° C. All slides were fixed in RiboFix, counterstained with 4′-6′diamidino-2-phenylindole (DAPI) using an antifade reagent (Ventana). Imaging was performed using the Nikon A1RVAAS laser point- and resonant-scanning confocal microscope equipped with a single photon Argon-ion laser at 40× with 4× zoom.
The 5 um cut paraffin sections and a prostate tissue array (Biomax us, PR8011 tissue array) were placed on Ventana's Discovery XT platform (Ventana Medical Systems, Inc., Tucson, Ariz.) for Chromogenic in-situ Hybridization (CISH). The deparaffinization of the sections was performed by the protocol that was selected on the instrument. All subsequent pretreatment steps were performed on the Ventana platform using FISH protocol and Ventana specific products. Offline detection staining was accomplished by Alkaline Phosphatase technique using Fast Red as chromogen. The custom made LNA probe with a dual FAM label from Exiqon was used during the denaturing and hybridizing steps and was incubated for 4 hours at the probe's optimal temperature for annealing. Three separate temperature controlled stringency washes were performed to wash away probe that was loosely bond. The primary rabbit anti-fluorescein antibody at a 1:100 dilution was applied with heat for 1 hour followed by Ventana's UltraMap anti-Rabbit-Alk Phos multimer detection for 20 mins no heat. The chromogenic detection was performed offline using the components of the Ventana ChromoRed kit. Slides were dehydrated and coverslipped to complete the protocol.
107 LNCaP cells were plated into 2 75-cm2 flasks and treated with either 10 ug/mL 5-aza-2′-deoxycytidine or left untreated. For 5 days, the cells were washed with PBS, fed fresh medium, and treated as above. After the fifth day all cells were washed with PBS, trypsinized, and centrifuged at 1200 rpm for 5′. The cell pellets were washed once with PBS, and purified using the QiaAmp DNA mini kit (QIAGEN). The samples were then quantified using the NanoDropND-100 spectrometer (Thermo scientific, Wilminton, Del., USA). 500 ng of genomic DNA was selected from each sample and treated with sodium bisulfite using the EZ DNA GOLD methylation kit (Zymo Research), eluting in 10 uL elution buffer.
PCR Amplification and Sequencing of Products Acquired from Bisulfite-Converted LNCAP Genomic DNA
50 ng of bisulfite-treated genomic DNA was used for bisulfite PCR using the following primer combination: 5′ Distal SPRY4 For (ggttttatttatttatttggttagtttt) and 5′ Distal SPRY4 Rev (taaatatcctttctctatcccaatc) to produce a 139-bp product. PCR was performed using a 2-min hot start at 95° C., followed by 40 cycles at 94° C. for 30 s, 48° C. for 35 s, and 72° C. for 30 s, ending with a 10-min extension at 72° C. using GoTaq green (Promega, Inc.). PCR products were run out on a 1% agarose gel, gel purified using the QiaQuick gel extraction kit (QIAGEN), and cloned into pCR4-TOPO (Invitrogen/Life Technologies). Six clones for each sample were sequenced using M13 forward and reverse primers (Retrogen, Inc.) and the results were aligned using VectorNTi AlignX (Invitrogen/Life Technologies).
Knock-down of SPRY4-IT1 was performed using a 25-mer double-stranded RNA oligonucleotide complex siRNA (gctttctgattccaaggcctattaa, labeled #594, Khaitan et al, 2011) and transfected into cells using lipofectamine RNAiMax (Life Technologies) in 6-well plates using manufacturer's protocols. A total of 250,000 cells were aliqouted into each well and the RNAi duplex-lipofectamine RNAiMAX complexes were added and mixed gently by rocking the plate. In all cases, cells were incubated for 48 hours at 37° C. in a CO2 incubator. Cell samples and gene expression levels were measured by quantitative real-time PCR (qRT-PCR, as above).
The MTT (3-(4,5-dimethyl-2-yl)-2,5-diphenyl-211-tetrazolium bromide) assay was purchased from Roche. 96-well plates were used, plating 25000 cells in 100 uL DMEM per well (transfected as above). 48 hours after of transfection, 20 uL MTT solution was added and the cells were incubated at 37° C. in the dark for 4 hours. Generated formazan was measured at OD490 nm to and compared to control cells to determine the cell viability on the Flex station (Molecular Devices; www.moleculardevices.com).
The invasion assay was performed using BD BioCoat™ growth factor reduced insert plates (Matrigel™ Invasion Chamber 12 well plates). These plates were prepared by rehydration of the BD Matrigel™ matrix coating and its inserts with 0.5 ml of serum-free DMEM media for 2 hours at 37° C. The media was removed from the inserts and 0.75 mL DMEM w/10% FBS was added to the lower chamber of the plate, with 0.5 mL of cell suspension (5×104 cells, transfected as above, in serum-free DMEM) added to each insert well. The invasion assay plates were then incubated for 48 hours at 37° C. After incubation, the non-invading cells were scrubbed from the upper surface of the insert. The cells on the bottom surface of the membrane were fixed in methanol, then stained with crystal violet, and washed in MQ H2O. The membranes were then mounted on microscopic slide for visualization and analysis. All slides were scanned (using the Scanscope digital slide scanner) and the number of cells remaining on the insert were counted using Aperio software. All data are expressed as the percent (%) invasion through the membrane versus the migration through the control membrane.
PC3 cells were plated in 96-well plates at 5000, 10000, & 15000 cells per well in triplicate for each transfection condition (transfected as above) and allowed to culture in DMEM w/10% FBS for 48 hours before harvesting for assay. Samples were then prepared using the Caspase-Glo® 3/7 Assay kit (Promega) and analyzed by a GloMax luminometer (Promega) using conditions designed for the Caspase-Glo 3/7 Assay.
This study included 18 pairs of formalin-fixed paraffin-embedded (FFPE) blocks of the prostate cancer and adjacent normal tissues. For the microarray experiments, 10 paired biopsy specimens were used for preparing RNA samples. These tissue samples were collected at Florida Hospital Celebration (Celebration, FL, USA) in 2008-2012. The use of tumor samples was approved by the institutional review board of the Florida hospital.
Twenty consecutive 18 um sections were cut from each patient block on a Leica 2235 microtome (Leica 2235) and placed into 2.0 ml microcentrifuge tubes. RNA was extracted with an RNeasy FFPE kit (QAIGEN). RNA yield and A260/A280 ratio were monitored with a NanoDropND-100 spectrometer (Thermo scientific, Wilminton, Del., USA). All qRT-PCR conditions performed were as in above methods. Fold changes in SPRY4-IT1 and SPRY4 expression in tumor tissue relative to the expression in normal tissue were calculated.
Urine samples were collected (30˜50 mL) using Urine Collection and Preservation Tube (Norgen Bioteck, Thorold, ON, Canada) and stored at −80° C. till further analysis. Total RNA was isolated using the Urine (Exfoliated cell) RNA Purification Kit (Norgen Bioteck, Thorold, ON, Canada). The purified RNA was quantified using the NanoDropND-100 spectrometer (Thermo scientific, Wilminton, Del., USA) and stored at −80° C. till further analysis.
RNA (100 ng) was used for cDNA synthesis in a 50 uL reaction volume using a high capacity cDNA reverse transcription kit (Applied Biosystems, Foster city, CA, USA). 5 ng of cDNA was used for pre-amplification in a 50 ul reaction volume containing 25 ul of 2× Power SYBR Green PCR master mix and 10 nM of each primer. The reaction was subjected to denaturation at 95° C. for 10 minutes followed by 14 cycles of denaturation at 95° C. for 15 seconds and annealing/elongation at 60° C. for 4 minutes.
qRT-PCR was performed in triplicate using a Power SYBR Green PCR master mix (Applied Biosystems, Warrington, UK) in the 7500 Real-Time PCR system (Applied Biosystems, Foster city, CA, USA). A final reaction volume of 20 ul was used, containing 1.14 ul of pre-amplified cDNA template, 10 ul of 2× Power SYBR Green PCR master mix (Applied Biosystems, Foster city, CA, USA), and 0.2 uM of each primer. The reaction was subjected to denaturation at 95° C. for 10 minute followed by 40 cycles of denaturation at 95° C. for 15 seconds and annealing at 58° C. for 1 minute. SDS1.2.3 software (Applied Biosystems, Foster city, CA, USA) was used for comparative Ct analysis with GAPDH serving as the endogenous control.
Putative prostate biomarker expression in urine samples was examined. Expression of eight lncRNAs (SPRY4-IT1, XLOC-007697, LOC100506411, LOC100287482, XLOC-009911, XLOC-008559, XLOC-005327, and XLOC-001699) and PCA3 was measured by qRT-PCR in one normal and three prostate cancer patients as shown in
The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.
The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
TTCAGGAAAGAGAAAATTATTCCTATCATCGGGGTTTTTGAAGAACATGAAATGA
GTGGTTGCTACCTTTAATCAGTACTATGGATTTCTAAATGCATTTAACTGTGGTTA
ATACTTTCAATGTGCATATTTTCATAATTTCATAATAAAGTTATCAATAAAAATA
AATAAAGAAGTGAAATTGCAATTCAAATAA
AAGATTTTGATTTACTAATTTATAATCTTATTTCCAAGCAAAACAAGTCAATTTCA
GCTGCACAATCTTTGAAGTGTAAGTTAATTTTTATGTGATATTTCAGTATATATTT
CGAGCAAATAAATATGCATTTCCCAGTGAAAAAAAAAAAAAAAAAAAAAAAAA
CAAGTTGAGGAACAGCCTATAAAATAACTGGC
GAACACCGAGAATAGCGTCATGTCATAAGGACTCAGAGCAGGTGGACCCTGCTG
GTCTTGTTTAAGTGATTTTCTTATAGTTTAAGAAATATATTGTGGTTTTGACCTTA
ATGCTTCAGCAACATTAGATGTTCTGGAGACTGGAAAGTCCAAGATCATGGTGCC
TTAAAAGGTACAATTCACAAGGTTGGAGGGGTAGCTGGAAGTTTCTGTGGTTACC
TTGCACTGGGGGGCTGCCCTGCCTCCACTCTCTCCCCACAGTCCGAGGGCAAGAT
AATAAAAACGTGTTTGTTTACTAAGTAA
GCTGACAGATTAACCCAACAAAGAAAGATTAGGAAAACAGCCTCAGCAATTTCC
ACTGAAATGGAAGATCCTGCTGTGAAAGGAGCAGTACAAAGAAAGAATGTACAG
ATCATGTTAGAACACTGTCTAGGAATGGTTGG
CTTCCTCTGTTATGCCAGATATGGTTAGCCACTTTGGTTTTTTAGGAGCTATAGGA
TGGGAAAAGCCTGAGTAATTCCTACACAGTGTGCTGAAATTAATAGAACTTTCAG
TTTGGTTCCATTTTTGTATCTCAGCTTCCAGGAAATAAAAAAGAATTCTAACATTC
GGAAAAAATGTTAATTACTGCAAATGTGTTTAAAACTGTAAAAGTACATTAAAC
AAAAGGTGAATAAAATCAGGTCACTCTTCT
CCATGCACTGTTAAATTTGATTTCAAGAAATTACAGGAAAACTTTCCAAAGTTCC
TTCTCCATGAGCTCAATAAAGTTTTTCAGGAACTCGG
AACTTTACATTCTTTACGGTTAAGCAAGATGTACAGCTCAGTCAAAGACACTAAA
TTTTCTGGTTCCAAACCAGATTTCCTGTGATTCTATACTAATAATTTTTGATATAA
AGGCCTATTAAAATTTCTGAGCATTGCCCATTTCTTTTGCTTTATCTGTAGGACAT
CTGTAGGATGTATATAGTTTAGGGGATTTTTTTTTTGTTTGGTTTTGTTTTTTAGAA
CTCCTTACTGGGTTTATTATAAGTGTCACATGTTTTTTATAATAAAACATAGGTGA
TGAATAATTTATTGTATTTTTAATTTGAATGTTTGTGCTTTTTAAATGAGCCAAGA
AAGTAGCTAGAGCCATGGAAGTACAGTATGAATTAAAAAGAAAAAAGTATTGAA
CTACA
GGCTCCTTATGTGCCTGAAAGAGTTTGAGTTTCCTGTTAACTCCAAATCAACAGT
TTCAGGAAAGAGAAAATTATTCCTATCATCGGGGTTTTTGAAGAACATGAAATGA
GTGGTTGCTACCTTTAATCAGTACTATGGATTTCTAAATGCATTTAACTGTGGTTA
ATACTTTCAATGTGCATATTTTCATAATTTCATAATAAAGTTATCAATAAAAATA
AATAAAGAAGTGAAATTGCAATTCAAATAA
AAGATTTTGATTTACTAATTTATAATCTTATTTCCAAGCAAAACAAGTCAATTTCA
GCTGCACAATCTTTGAAGTGTAAGTTAATTTTTATGTGATATTTCAGTATATATTT
CGAGCAAATAAATATGCATTTCCCAGTGAAAAAAAAAAAAAAAAAAAAAAAAA
CAAGTTGAGGAACAGCCTATAAAATAACTGGC
GAACACCGAGAATAGCGTCATGTCATAAGGACTCAGAGCAGGTGGACCCTGCTG
GTCTTGTTTAAGTGATTTTCTTATAGTTTAAGAAATATATTGTGGTTTTGACCTTA
TGGTA
TTAAAAGGTACAATTCACAAGGTTGGAGGGGTAGCTGGAAGTTTCTGTGGTTACC
TTGCACTGGGGGGCTGCCCTGCCTCCACTCTCTCCCCACAGTCCGAGGGCAAGAT
AATAAAAACGTGTTTGTTTACTAAGTAA
CAACAAAGAAAGATTGGGAAAACATAACCTCAGCAATTTCCCAAGAAACTGAAG
ACTGAAATGGAAGATCCTGCTGTGAAAGGAGCAGTACAAAGAAAGAATGTACAG
ATCATGTTAGAACACTGTCTAGGAATGGTTGG
CTTCCTCTGTTATGCCAGATATGGTTAGCCACTTTGGTTTTTTAGGAGCTATAGGA
TGGGAAAAGCCTGAGTAATTCCTACACAGTGTGCTGAAATTAATAGAACTTTCAG
TTTGGTTCCATTTTTGTATCTCAGCTTCCAGGAAATAAAAAAGAATTCTAACATTC
GGAAAAAATGTTAATTACTGCAAATGTGTTTAAAACTGTAAAAGTACATTAAAC
AAAAGGTGAATAAAATCAGGTCACTCTTCT
CCATGCACTGTTAAATTTGATTTCAAGAAATTACAGGAAAACTTTCCAAAGTTCC
TTCTCCATGAGCTCAATAAAGTTTTTCAGGAACTCGG
AACTTTACATTCTTTACGGTTAAGCAAGATGTACAGCTCAGTCAAAGACACTAAA
TTTTCTGGTTCCAAACCAGATTTCCTGTGATTCTATACTAATAATTTTTGATATAA
AGGCCTATTAAAATTTCTGAGCATTGCCCATTTCTTTTGCTTTATCTGTAGGACAT
CTGTAGGATGTATATAGTTTAGGGGATTTTTTTTTTGTTTGGTTTTGTTTTTTAGAA
CTCCTTACTGGGTTTATTATAAGTGTCACATGTTTTTTATAATAAAACATAGGTGA
TGAATAATTTATTGTATTTTTAATTTGAATGTTTGTGCTTTTTAAATGAGCCAAGA
AAGTAGCTAGAGCCATGGAAGTACAGTATGAATTAAAAAGAAAAAAGTATTGAA
CTACA
GGCTCCTTATGTGCCTGAAAGAGTTTGAGTTTCCTGTTAACTCCAAATCAACAGT
ATTGAAAGGGTTTCTGTGAATACCTCCACAACTGTGGTTCTCAAAGTGTAATCCC
AAAAGGCAAGAAATCAGCAAGAGAGAGAGATGAAGCATGAGAAATGAGCAAAA
AGAGAAGAGAGAACGGCTTACAGCTCAGGTCCTCTCTCCATGCTTAGGAACCAC
GAAGGAGGAGAGAGAGAGATTTTATAAAAGGCATGATCCATGAAAGAAAG
GAGAGAGATTTGACTGGGCTAAGAAAGAAATGAACACGATTT
TGTTCTTTTCTTATAAATTTATAGTTGTATTTATTCTGAAGTTCTTATCTGAGTTGA
ATCTGGAGCTTAGATGGAGAGAGAAGAGAGAGATTAATTGAGGCCCCAGGTACT
GAATAAACTTGAACATTCTGACTTTGAAGAACATGACCAGGCTAGCCCAGGAGA
AATAAGTCTGTAGGCCTTGTCTGTTAATAAATAGTTTATATACCAAAAAAAAAAA
CAGCTGCCAACAAGAAGCATTGGAACAAACCATGCTGGGTTAATACAT
This application claims priority to and benefit of U.S. Provisional Patent Application Ser. No. 61/909,319, filed Nov. 26, 2013 and U.S. Provisional Patent Application Ser. No. 61/920,318, filed Dec. 23, 2013, the disclosures of which are incorporated herein by reference in their entirety for all purposes.
Filing Document | Filing Date | Country | Kind |
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PCT/US2014/067747 | 11/26/2014 | WO | 00 |
Number | Date | Country | |
---|---|---|---|
61909319 | Nov 2013 | US | |
61920318 | Dec 2013 | US |