Oncogenic Activity of miR-650 in Prostate Cancer Is Mediated by Suppression of CSR1 Expression
2015; Elsevier BV; Volume: 185; Issue: 7 Linguagem: Inglês
10.1016/j.ajpath.2015.03.015
ISSN1525-2191
AutoresZehua Zuo, Yan Yu, Ying Ding, Silvia Liu, Amantha Martin, George C. Tseng, Jianhua Luo,
Tópico(s)RNA Research and Splicing
ResumoCellular stress response 1 (CSR1) is a tumor suppressor gene whose expression was frequently down-regulated in prostate cancer. The mechanism of its down-regulation, however, is not clear. Here, we show that the 3′ untranslated region of CSR1 contains a target site of miR-650. High level of miR-650 was found in prostate cancer samples and cell lines. Degradation of miR-650 by specific inhibitor dramatically increased the expression levels of CSR1. Interaction between miR-650 and its target site in the 3′ untranslated region was validated through luciferase reporter system. Mutation at the target site completely abrogated the activity of miR-650 on the 3′ untranslated region of CSR1. Inhibition of miR-650 reversed the expression suppression of CSR1, suppressed colony formation, and blocked cell cycle entry to the S phase of both PC3 and DU145 cells. Animal model showed significant decrease of tumor volume, rate of metastasis, and mortality of severe combined immunodeficient mice xenografted with PC3 or DU145 cells transformed with inhibitor of miR-650. Our analyses demonstrate that suppression of CSR1 expression is a novel mechanism critical for the oncogenic activity of miR-650. Cellular stress response 1 (CSR1) is a tumor suppressor gene whose expression was frequently down-regulated in prostate cancer. The mechanism of its down-regulation, however, is not clear. Here, we show that the 3′ untranslated region of CSR1 contains a target site of miR-650. High level of miR-650 was found in prostate cancer samples and cell lines. Degradation of miR-650 by specific inhibitor dramatically increased the expression levels of CSR1. Interaction between miR-650 and its target site in the 3′ untranslated region was validated through luciferase reporter system. Mutation at the target site completely abrogated the activity of miR-650 on the 3′ untranslated region of CSR1. Inhibition of miR-650 reversed the expression suppression of CSR1, suppressed colony formation, and blocked cell cycle entry to the S phase of both PC3 and DU145 cells. Animal model showed significant decrease of tumor volume, rate of metastasis, and mortality of severe combined immunodeficient mice xenografted with PC3 or DU145 cells transformed with inhibitor of miR-650. Our analyses demonstrate that suppression of CSR1 expression is a novel mechanism critical for the oncogenic activity of miR-650. miRNAs are some well-conserved small noncoding RNA molecules of 20 to 22 bases in plants and mammals, playing a critical role in epigenetic regulation of gene expression.1Chen K. Rajewsky N. The evolution of gene regulation by transcription factors and microRNAs.Nat Rev Genet. 2007; 8: 93-103Crossref PubMed Scopus (1202) Google Scholar miRNAs silence targeted mRNA through base-pairing with the complementary sequences within the 3′ untranslated regions (UTRs) of mRNA molecules and block the ribosome complexes from translating the mRNAs into proteins.2Bartel D.P. MicroRNAs: target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (15850) Google Scholar Approximately 1000 miRNAs are present in the human genome and are projected to target up to 60% of mRNA species.3Bentwich I. Avniel A. Karov Y. Aharonov R. Gilad S. Barad O. Barzilai A. Einat P. Einav U. Meiri E. Sharon E. Spector Y. Bentwich Z. Identification of hundreds of conserved and nonconserved human microRNAs.Nat Genet. 2005; 37: 766-770Crossref PubMed Scopus (1561) Google Scholar, 4Lewis B.P. Burge C.B. Bartel D.P. 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Capece D. Zazzeroni F. Alesse E. MicroRNAs in the DNA damage/repair network and cancer.Int J Genomics. 2014; 2014: 820248Crossref PubMed Scopus (67) Google Scholar, 7Zhang W. Zhang J. Hoadley K. Kushwaha D. Ramakrishnan V. Li S. Kang C. You Y. Jiang C. Song S.W. Jiang T. Chen C.C. miR-181d: a predictive glioblastoma biomarker that downregulates MGMT expression.Neuro Oncol. 2012; 14: 712-719Crossref PubMed Scopus (147) Google Scholar Abnormal expression of miR-650 was previously shown to be associated with melanoma,8Chan E. Patel R. Nallur S. Ratner E. Bacchiocchi A. Hoyt K. Szpakowski S. Godshalk S. Ariyan S. Sznol M. Halaban R. Krauthammer M. Tuck D. Slack F.J. Weidhaas J.B. MicroRNA signatures differentiate melanoma subtypes.Cell Cycle. 2011; 10: 1845-1852Crossref PubMed Scopus (82) Google Scholar gastric cancer,9Zhang X. Zhu W. Zhang J. Huo S. Zhou L. Gu Z. Zhang M. MicroRNA-650 targets ING4 to promote gastric cancer tumorigenicity.Biochem Biophys Res Commun. 2010; 395: 275-280Crossref PubMed Scopus (108) Google Scholar non-small cell lung cancer,10Huang J.Y. Cui S.Y. Chen Y.T. Song H.Z. Huang G.C. Feng B. Sun M. De W. Wang R. Chen L.B. MicroRNA-650 was a prognostic factor in human lung adenocarcinoma and confers the docetaxel chemoresistance of lung adenocarcinoma cells via regulating Bcl-2/Bax expression.PLoS One. 2013; 8: e72615Crossref PubMed Scopus (62) Google Scholar chronic lymphocytic leukemia,11Mraz M. Dolezalova D. Plevova K. Stano Kozubik K. Mayerova V. Cerna K. Musilova K. Tichy B. Pavlova S. Borsky M. Verner J. Doubek M. Brychtova Y. Trbusek M. Hampl A. Mayer J. Pospisilova S. MicroRNA-650 expression is influenced by immunoglobulin gene rearrangement and affects the biology of chronic lymphocytic leukemia.Blood. 2012; 119: 2110-2113Crossref PubMed Scopus (82) Google Scholar and hepatocellular carcinoma.12Zeng Z.L. Li F.J. Gao F. Sun D.S. Yao L. Upregulation of miR-650 is correlated with down-regulation of ING4 and progression of hepatocellular carcinoma.J Surg Oncol. 2013; 107: 105-110Crossref PubMed Scopus (52) Google Scholar High level of miR-650 was shown to be a prognosticator for lymph node involvement and more aggressive clinical outcomes of lung adenocarcinoma. However, the mechanism of miR-650–induced tumorigenesis remains unclear. Here, we showed that miR-650 was significantly up-regulated in prostate cancer (PC). High expression level of miR-650 was significantly associated with lower prostate-specific antigen–free survival rate. Expression of miR-650 inversely correlated with that of cellular stress response 1 (CSR1), a tumor suppressor gene that is frequently down-regulated in PC. The oncogenic activity of miR-650 in PC is mediated by inhibition of CSR1 expression. All cell lines, including PC3 and DU145, were purchased from ATCC (Manassas, VA). PC3 cells were cultured with F12K medium supplemented with 10% fetal bovine serum (Invitrogen, Carlsbad, CA). DU145 cells were cultured with modified Eagle medium supplemented with 10% fetal bovine serum (Invitrogen). These cell lines were purchased from ATCC in 2011. Authentication with short tandem repeat profiling was performed in 2013. CSR1 antisera were raised in rabbits immunized with peptides that corresponded to CSR1 coding sequence regions (AGLDLSLKDLTQECYDVKAAVHQINF). These antisera were peptide affinity-purified with the AminoLink kit from Pierce (Rockford, IL). The purified antisera were tested for binding specificity for CSR1 in a Western blot analysis with protein extracts from cells overexpressing CSR1 (PDC1 and PDC4)13Yu G. Tseng G.C. Yu Y.P. Gavel T. Nelson J. Wells A. Michalopoulos G. Kokkinakis D. Luo J.H. CSR1 suppresses tumor growth and metastasis of prostate cancer.Am J Pathol. 2006; 168: 597-607Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar or cells not expressing CSR1 (PC3 and LNCaP cells). The antisera detected a single band of 72-kd protein (the predicted molecular weight of CSR1). The PC specimens that were analyzed were archived as frozen or formalin-fixed, paraffin-embedded specimens of tissues from radical prostatectomies from 1985 through 2007. Specimens were selected largely on the basis of their availability or whether sufficient amounts of tumor tissues were present. The ages of patients at the time of surgery ranged from 45 through 79 years. For formalin-fixed, paraffin-embedded samples, 617 samples were collected, including 77 organ donor (OD) prostates from patients free of urologic diseases (obtained while patients were on heart-lung machine support), 324 benign prostate tissues adjacent to cancer, and 216 PCs. Two hundred fifteen of the 216 corresponding patients were followed clinically for at least 5 years. In addition, frozen tissues from 11 ODs, 20 benign prostate tissues adjacent to cancer, and 22 PC samples were obtained for quantitative real-time RT-PCR (RT-qPCR). The protocols were approved by the University of Pittsburgh Institutional Review Board (Pittsburgh, PA). Pure tumor specimens were obtained by dissecting freshly resected tissues, typically within 30 minutes of removal from patients. These tissues were frozen at −80°C and were selected on the basis of tissue availability. Tissues were retrieved and microdissected immediately to 75% purity through microscopic needle dissection before the extraction of DNA or total RNA. Tumor cells were microdissected from frozen sections on slides by board-certified pathologists. For tissue microarray analysis, 671 formalin-fixed, paraffin-embedded prostate tissue specimens were arrayed onto six slides, with one or two samples from each specimen.14Ren B. Yu G. Tseng G.C. Cieply K. Gavel T. Nelson J. Michalopoulos G. Yu Y.P. Luo J.H. MCM7 amplification and overexpression are associated with prostate cancer progression.Oncogene. 2006; 25: 1090-1098Crossref PubMed Scopus (148) Google Scholar Patients in this group ranged in age from 45 to 79 years, and complete 5-year follow-up data were available for 215 patients with PC (University of Pittsburgh Medical Center tissue collection archive, 1985 through 2000). Immunohistochemistry was performed as described previously15Jing L. Liu L. Yu Y.P. Dhir R. Acquafondada M. Landsittel D. Cieply K. Wells A. Luo J.H. Expression of myopodin induces suppression of tumor growth and metastasis.Am J Pathol. 2004; 164: 1799-1806Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar with purified CSR1 peptide antiserum (dilution 1:1000). Antigen retrieval was performed with 25 mmol/L sodium citrate buffer (pH 9.0) at 90°C for 15 minutes, followed by treatment of 3% H2O2 to block endogenous peroxidase. The slides were incubated at room temperature for 2 hours with anti-CSR1 antibodies at a 1:400 dilution. The sections were then incubated with horseradish peroxidase–conjugated anti-rabbit IgG for 30 minutes at room temperature. For visualization, horseradish peroxidase was reacted with 3,3′-diaminobenzidine solution (Dako, Carpinteria, CA). Cells were counterstained with hematoxylin. Immunohistochemistry specificity was verified by incubating similar slides with preimmune sera. The results of immunostaining were graded by three independent observers as 0 to 3 on the basis of intensity, with 0 being negative, 0.5 being focal positive, 1 being weakly positive, 2 being moderately positive, and 3 being strong positive. Each score represents the average value of five high-power fields. The procedure of in situ hybridization was previously described.16Luo J.H. Yu Y.P. Cieply K. Lin F. Deflavia P. Dhir R. Finkelstein S. Michalopoulos G. Becich M. Gene expression analysis of prostate cancers.Mol Carcinog. 2002; 33: 25-35Crossref PubMed Scopus (210) Google Scholar Briefly, formalin-fixed, paraffin-embedded tissue slides were treated with xylene for 16 hours, followed by washing in phosphate-buffered saline at room temperature three times. The tissues were then treated with proteinase K at 37°C for 15 minutes and washed with phosphate-buffered saline. The slides were quenched with 0.1% H2O2 for 20 minutes and washed with phosphate-buffered saline. Hybridization was performed with a cocktail of biotin-cytosin– and bio-guanine–labeled oligonucleotides (5′-RGRURCRCRURGRARGRARGRCRGRCRURGRCRCRURCRCRU-3′) in 6× standard saline citrate complementary to miR-650 sequence, which was added to the slides for 16 hours at 37°C. The nonhybridized probes were removed by washing the tissues sequentially with 4× standard saline citrate, 2× standard saline citrate, and 0.2× standard saline citrate. The signals of hybridization were then amplified with one cycle of streptoavidin and biotinylated anti-streptoavidin treatment (ABC kit; Vector Laboratories, Burlingame, CA). The results of in situ hybridization were then visualized with diaminobenzidin treatment of the slides. The negative controls received a scramble biotin-labeled oligo probe (5′-TCCTGCGAGGCTGGTGGCTCCTGC-3′). The results of in situ hybridization were graded by three independent observers as 0 to 3 on the basis of intensity, with 0 being negative, 0.5 being focal positive, 1 being weakly positive, 2 being moderately positive, and 3 being strong positive. Each score represents the average value of five high-power fields. CSR1 expression in PC3 or DU145 cells transformed with pSingle-tTs-InhniR650 or pSingle-tTs-mimic miRNA650 was examined. First, cells were washed with phosphate-buffered saline and lyzed by RIPA buffer (50 mmol/L Tris-HCl at pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonyl fluoride, aprotinin at 1 μg/mL, leupeptin at 1 μg/mL, pepstatin at 1 μg/mL, and 1 mmol/L Na3VO4). The lysates were sonicated and centrifuged at 12,000 × g at 4°C for 30 minutes to remove the insoluble materials. The proteins were separated by SDS-PAGE in 8.5% polyacrylamide gels and were transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% powdered skim milk in Tris–Tween 20 buffer (0.1 mol/L Tris-HCl and 0.1% Tween 20, pH 7.4) for 1 hour at room temperature, followed by a 2-hour incubation with primary anti-CSR1 antibodies (dilution 1:1000) or anti–glyceraldehyde-3-phosphate dehydrogenase antibodies (dilution 1:500; Abcam Inc., Cambridge, MA). The membrane was then washed three times with Tris-Tween 20 buffer and incubated with a horseradish peroxidase–conjugated secondary antibody specific for rabbit (anti-CSR1; dilution 1:1000) or goat (anti–glyceraldehyde-3-phosphate dehydrogenase; dilution 1:1000) for 1 hour at room temperature. The protein expression was detected with the electrochemiluminescence system (Amersham Life Science, Piscataway, NJ) according to the manufacturer's protocols. Colony formation assays were similar to those previously described.15Jing L. Liu L. Yu Y.P. Dhir R. Acquafondada M. Landsittel D. Cieply K. Wells A. Luo J.H. Expression of myopodin induces suppression of tumor growth and metastasis.Am J Pathol. 2004; 164: 1799-1806Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 17Zheng Z.L. Tan L.Z. Yu Y.P. Michalopoulos G. Luo J.H. Interaction of CSR1 with XIAP reverses inhibition of caspases and accelerates cell death.Am J Pathol. 2012; 181: 463-471Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 18Wang H. Luo K. Tan L.Z. Ren B.G. Gu L.Q. Michalopoulos G. Luo J.H. Yu Y.P. p53-induced gene 3 mediates cell death induced by glutathione peroxidase 3.J Biol Chem. 2012; 287: 16890-16902Crossref PubMed Scopus (48) Google Scholar, 19Han Y.C. Yu Y.P. Nelson J. Wu C. Wang H. Michalopoulos G.K. Luo J.H. Interaction of integrin-linked kinase and miniature chromosome maintenance 7-mediating integrin {alpha}7 induced cell growth suppression.Cancer Res. 2010; 70: 4375-4384Crossref PubMed Scopus (24) Google Scholar, 20Zhu Z.H. Yu Y.P. Shi Y.K. Nelson J.B. Luo J.H. CSR1 induces cell death through inactivation of CPSF3.Oncogene. 2009; 28: 41-51Crossref PubMed Scopus (35) Google Scholar, 21Yu Y.P. Yu G. Tseng G. Cieply K. Nelson J. Defrances M. Zarnegar R. Michalopoulos G. Luo J.H. Glutathione peroxidase 3, deleted or methylated in prostate cancer, suppresses prostate cancer growth and metastasis.Cancer Res. 2007; 67: 8043-8050Crossref PubMed Scopus (177) Google Scholar, 22Ren B. Yu Y.P. Tseng G.C. Wu C. Chen K. Rao U.N. Nelson J. Michalopoulos G.K. Luo J.H. Analysis of integrin alpha7 mutations in prostate cancer, liver cancer, glioblastoma multiforme, and leiomyosarcoma.J Natl Cancer Inst. 2007; 99: 868-880Crossref PubMed Scopus (56) Google Scholar PIN 6 and DIN 8 cells were used. For colony formation assay, 5000 cells were cultured in 60-mm dishes. Triplicate experiments were performed for each cell clones. Medium was changed every 4 days. On the 10th day, the plates were stained with 1% crystal violet, and colonies with a diameter >2 mm were counted. To construct the vector that expresses inhibitor and mimics of miR-650 sequence, the following oligonucleotides were used: 5′-TCGAGGTCCTGAGAGCGCTGCCTCCTTTTTTTACGCGT-3′ and 5′-AGCTTACGCGTAAAAAAAGGAGGCAGCGCtCtCAGGACC-3′ for inhibitor of miR-650, 5′-TCGAGGTCCTGAGAGCGCTGCCTCCTTTTTTTACGCGT-3′ and 5′-AGCTTACGCGTAAAAAAAGGAGGCAGCGCTCTCAGGACC-3′ for mimics of miR-650. These primers were annealed and ligated into a pSingle-tTs-shRNA vector. The ligated products were transfected into Escherichia coli and plated on kanamycin plates (50 μg/mL). Fifteen colonies per transfection were picked and sequenced for the presence of inserts. The selected clones, which suppressed the expression of miR-650, were then transfected into PC3 and DU145 cells to generate cells that are doxycycline inducible for the suppression of miR-650. Induction of inhibitor of miR-650 was verified by RT-qPCR of cells induced with 5 μg/mL doxycycline with the use of the following primers: 5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAGGAGG-3′ and 5′-CGGCGTCCTGAGAGCGCTG-3′. To generate pLuc-CSR1, PCR was performed with primers 5′-CGGAAGACAGACGAGGAGAC-3′ and 5′-GTCATCCAGGAAGGACGAGA-3′ on CSR1 cDNA template under the following conditions: 94°C for 5 minutes then 30 cycles of 94°C for 30 seconds, 63°C for 1 minute, and 72°C for 4 minutes. This was followed by extension at 72°C for 10 minutes. For mutant pLuc-CSR1, separate PCRs were performed on CSR1 cDNA template with the use of primer sets 5′-GTTCTCTCTGCACAGCTTTTTCCCTTGCTCTCCCTG-3′ and 5′-ATCATGTCTGCTCGAAGCGG-3′ and 5′-GGACTGACCGGCAAGTTGGAC-3′ and 5′-CAGGGAGAGCAAGGGAAAAAGCTGTGCAGAGAGAAC-3′. Second PCR was then performed on purified combined PCR products from the first PCRs by using primers 5′-GGACTGACCGGCAAGTTGGAC-3′ and 5′-ATCATGTCTGCTCGAAGCGG-3′. The PCR products were then ligated into pLuc vector. The transfection efficiency was examined by co-transfection of cyanine 5 5′ end-labeled scramble oligonucleotide sequence 5′-TAATGTATTGGAACGCATATT-3′. Approximately 1 × 107 viable PIN 6 and DIN 8 cells, suspended in 0.2 mL of Hanks' balanced salt solution (Krackeler Scientific, Inc., Albany, NY), were subcutaneously implanted in the abdominal flanks of 32 severe combined immunodeficient mice to generate one tumor per mouse. Mice were observed daily, and their body weight, tumor size, and lymph node enlargement were recorded weekly. Tumor and lymph node sizes were measured on the diameter. After 6 weeks or when mice became moribund, whichever occurred first, the mice were sacrificed, and necropsies were performed. Serial sections of formalin-fixed, paraffin-embedded lung, brain, liver, kidney, vertebra, and lymph node specimens were collected, stained with hematoxylin and eosin, and examined microscopically. All animal procedures were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Standard errors for individual proportions were calculated by use of the exact binomial test, and those for a numerical distribution were calculated with the following conventional independent and normal assumption: means ± (1.96 × SD/n1/2), where n = sample size.23Rice J. Mathematical Statistics and Data Analysis. Duxbury Press, Pacific Grove, CA2006Google Scholar Comparison of two proportions was inferred by Fisher's exact test because of the relatively small sample sizes (function fisher.test in R package).23Rice J. Mathematical Statistics and Data Analysis. Duxbury Press, Pacific Grove, CA2006Google Scholar Survival was analyzed by the Kaplan-Meier method, and survival curves were compared by use of the log-rank test.24Hosmer D.W. Lemeshow S. Applied Survival Analysis. Wiley, Hoboken, NJ2003Google Scholar All statistical tests were two-sided. The mechanisms that govern the expression of CSR1 remain unclear. To investigate the potential mechanism of CSR1 expression regulation, the 3′ UTR of CSR1 was analyzed for miRNA targeting by using miRWalk version 2.0 (http://www.umm.uni-heidelberg.de/apps/zmf/mirwalk, last accessed March 5, 2015). A total of 20 potential miRNA target sites and 16 unique miRNAs were identified targeting at the 3′ UTR of CSR1. Among these miRNAs, five are known to be oncogenic (miR-297, -484, -1182, -210, and -650). To investigate the relevance of these oncogenic miRNAs in PC, RT-qPCRs with primers corresponding to these miRNAs were performed on PC cell lines PC3, DU145, and LNCaP and 11 fresh-frozen OD prostate samples. Only miR-650 was shown to be up-regulated in PC cell lines (Figure 1A ). As a result, miR-650 was selected for further analyses. miR-650 was implicated in a number of human malignancies.8Chan E. Patel R. Nallur S. Ratner E. Bacchiocchi A. Hoyt K. Szpakowski S. Godshalk S. Ariyan S. Sznol M. Halaban R. Krauthammer M. Tuck D. Slack F.J. Weidhaas J.B. MicroRNA signatures differentiate melanoma subtypes.Cell Cycle. 2011; 10: 1845-1852Crossref PubMed Scopus (82) Google Scholar, 9Zhang X. Zhu W. Zhang J. Huo S. Zhou L. Gu Z. Zhang M. MicroRNA-650 targets ING4 to promote gastric cancer tumorigenicity.Biochem Biophys Res Commun. 2010; 395: 275-280Crossref PubMed Scopus (108) Google Scholar, 10Huang J.Y. Cui S.Y. Chen Y.T. Song H.Z. Huang G.C. Feng B. Sun M. De W. Wang R. Chen L.B. MicroRNA-650 was a prognostic factor in human lung adenocarcinoma and confers the docetaxel chemoresistance of lung adenocarcinoma cells via regulating Bcl-2/Bax expression.PLoS One. 2013; 8: e72615Crossref PubMed Scopus (62) Google Scholar, 11Mraz M. Dolezalova D. Plevova K. Stano Kozubik K. Mayerova V. Cerna K. Musilova K. Tichy B. Pavlova S. Borsky M. Verner J. Doubek M. Brychtova Y. Trbusek M. Hampl A. Mayer J. Pospisilova S. MicroRNA-650 expression is influenced by immunoglobulin gene rearrangement and affects the biology of chronic lymphocytic leukemia.Blood. 2012; 119: 2110-2113Crossref PubMed Scopus (82) Google Scholar, 12Zeng Z.L. Li F.J. Gao F. Sun D.S. Yao L. Upregulation of miR-650 is correlated with down-regulation of ING4 and progression of hepatocellular carcinoma.J Surg Oncol. 2013; 107: 105-110Crossref PubMed Scopus (52) Google Scholar To investigate the role of miR-650 in PC, RT-qPCR with primers specific for miR-650 was performed on 56 fresh-frozen prostate samples, including 22 PC, 20 benign prostate tissues adjacent to cancer (AT), 11 OD prostates, and three PC cell lines (PC3, DU145, and LNCaP). Our results show an average 2.2-fold increase of miR-650 in PC samples in comparison with benign prostate tissues adjacent to cancer, and more than eightfold increase over the OD prostates from patients free of urologic disease (Figure 1A). To investigate the clinical correlation between the expression of miR-650 and PC, in situ hybridizations of miR-650 were performed on 617 formalin-fixed, paraffin-embedded prostate samples, including 216 PC, 77 OD prostates, and 324 benign prostate tissues adjacent to cancer. The signals of hybridization from each sample were classified on the basis of intensity as strong positive (score = 3), moderate positive (score = 2), weakly positive (score = 1), focal (score = 0.5), or negative (score = 0). The score from each sample represents the average of scores graded by three observers. Most of the PC samples were at least weakly positive for miR-650 (average score = 1.14) (Figure 1B). In contrast, expressions of miR-650 were mostly focal in both OD (average score = 0.23) and benign prostate tissue adjacent to cancer (average score = 0.32) (Figure 1, B and C). This represents a 3.6-fold increase of miR-650 expression in PC cells in comparison with benign prostate tissues adjacent to cancer (P = 4.9 × 10−48) and a 4.9-fold in comparison with OD prostates (P = 1.3 × 10−43). The expression of miR-650 is significantly associated with higher (8 to 10 versus 5 to 7) Gleason's grade (P = 0.012) (Table 1). When PC samples were segregated on the basis of miR-650 score cutoff at 2, patients with miR-650 expression scores of at least 2 experienced significantly lower rate of prostate-specific antigen-free survival (31%) in comparison with patients with score <2 (86%; P = 4.4 × 10−16) (Figure 1D). These results suggest that expression of miR-650 correlates with the aggressiveness of PC and is indicative of a poor clinical outcome.Table 1Association of Gleason's Grade and miR-650 Expression ScoresGleason's gradeNo. of samplesmiR-650 score (means ± SD)520.75 ± 0.186851.15 ± 0.077580.96 ± 0.088411.3 ± 0.099211.25 ± 0.171091.38 ± 0.27Combined Gleason 5–71451.07 ± 0.05 8–10711.29 ± 0.08 Open table in a new tab Previous works had shown a significant down-regulation of CSR1 in PC and glioblastoma multiformis.13Yu G. Tseng G.C. Yu Y.P. Gavel T. Nelson J. Wells A. Michalopoulos G. Kokkinakis D. Luo J.H. CSR1 suppresses tumor growth and metastasis of prostate cancer.Am J Pathol. 2006; 168: 597-607Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar Expression of CSR1 inhibited PC growth and induced cell death. Thirteen bases of miR-650 appear to complement to a sequence in 3′ UTR in CSR1 (c.2996-3015 of NM_016240.2) (Figure 2A). We hypothesize that high level of expression of miR-650 correlates with low level of expression of CSR1. To examine whether there is an inverse correlation between miR-650 and CSR1 expression, 53 fresh-frozen prostate samples that were examined for miR-650 were evaluated for CSR1 expression with RT-qPCR. The results showed a trend of inverse correlation between CSR1 and miR-650 expressions (Pearson correlation coefficient = −0.76) (Figure 2B ). To validate this correlation, a correlation analysis was performed on a separate cohort of 166 formalin-fixed, paraffin-embedded samples of PC and benign prostate tissues with immunohistochemistry staining of CSR1 expression and in situ hybridization of miR-650. The correlation yielded a similar inverse correlation of miR-650 and CSR1 expression (Pearson correlation coefficient = −0.7) (Figure 2C). These results support an inverse relation between CSR1 and miR-650 expression. To examine whether the putative miR-650 target site in the 3′ UTR of CSR1 is functional, an inhibitor of miR-650 was constructed into pSingle-tTs-shRNA vector to generate pSingle-inhmiR-650. This vector was then transfected into PC3 and DU145 cells. Fifteen colonies from each lineage were selected. Two clones (PIN 6 from PC3 cells transformed with pSingle-inhmiR-650 and DIN 8 from DU145 cells transformed with pSingle-inhmiR-650) were selected for characterization. Our analyses showed that induction of PIN 6 and DIN 8 with doxycycline (5 μg/mL) reduced the expression of miR-650 by 8.2- and 4.8-fold in PIN 6 and DIN 8 cells, respectively (Figure 3A). The decrease of miR-650 expression was accompanied with 27- and 40-fold increase of CSR1 mRNA in these cells (Figure 3B). The increase of CSR1 mRNA resulted in dramatically higher level of CSR1 in these cells (Figure 3C). To investigate whether the putative miR-650 target site in the 3′ UTR of CSR1 is functional in a different context, the 3′ UTR sequence (1650 bp) of CSR1 was ligated into the 3′ end of luciferase reporter gene that is driven by phosphoglycerate kinase 1 promoter to generate pLuc-CSR1 (Figure 3D). Co-transfection of pLuc-CSR1 and pSingle-mimiR-650, a mimic of miR-650, reduced the reporter gene activity by 60% (P < 0.001) in comparison with the controls (Figure 3E). In contrast, when pLuc-CSR1 was co-transfected with pSingle-inhmiR-650, the reporter gene activity increased by 40% (P < 0.001). To investigate whether the putative target site is critical for miR-650 activity, a four-nucleotide point mutation was introduced to the miR-650 binding site in the 3′ UTR of CSR1 sequence. The mutation completely abrogated the activity of miR-650 (Figure 3E). These results indicate that the miR-650 target sequence in 3′ UTR is the crucial binding site for miR-650 and mediates its CSR1 down-regulation activity. CSR1 contains tumor suppressor activity. High level expression of miR-650 in PC cell lines suppresses CSR1 expression. Because down-regulation of CSR1 induces carcinogenesis, we hypothesize that miR-650 contains oncogenic activity because of its negative impact on CSR1 expression. When PIN 6 and DIN 8 cells were induced to express the inhibitor of miR-650, expression of CSR1 was recovered in both cell lines (Figure 4, A and B ). The higher levels of CSR1 were accompanied with >50% to 55% drops in colony formation in PIN 6 and DIN 8 cells. Cell cycle analyses indicate that expression of inhibitor of miR-65
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