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779 MICROARRAY ANALYSIS OF MIRNA EXPRESSION IN DIFFERENT PROSTATE CANCER CELL LINES
P47 GENETIC AND EPIGENETIC CHANGES IN PROSTATE CANCER Friday, 20 March, 12.15-13.45, Room C5
777
778
miR-15a functions as a tumor suppressor for prostate cancer by targeting multiple oncogenes
Upregulation of microRNA-34c affect proliferation, migration, invasion and apoptosis in prostate cells
Kojima S.1, Watanabe R.1, Tsukada J.2, Furuya Y.1, Seki N.2
Hagman Z.H., Ceder A.Y.C.
Teikyo University Chiba Medical Center, Dept. of Urology, Ichihara, Japan, 2Chiba University School of Medicine, Dept. of Functional Genomics, Chiba, Japan
Clinical Chemistry, Dept. of Laboratory Medicine, Malmö, Sweden
Introduction & Objectives: MicroRNAs (miRNAs) are short noncoding RNAs regulating gene expression that play roles in human cancer. Each miRNA is predicted to regulate hundreds of transcripts, but few have been experimentally validated. The miR-15a cluster resides at chromosome 13q14.3, a genomic region frequently deleted in human cancers including prostate cancer (PCa). Our previous works indicated that expression levels of miR-15a were down-regulated in PCa specimens. Here, to investigate the role of tumorigenesis of miR-15a as tumor suppressors in PCa. In addition, we subjected to target gene prediction of miR-15a using genome-wide gene expression analysis.
Introduction & Objectives: MicroRNA (miRNA) are naturally occurring short noncoding RNA (18-25 nt) that regulate gene expression by inhibiting translation of their target mRNA by non perfect base pairing to the 3’UTRs. To date, nearly 700 human miRNAs have been identified, but this is expected to increase to around 1000. Recently, miRNAs was suggested to be of relevance for cancer progression after discovering that the miRNA expression pattern change and that there are recurrent amplification and deletion of miRNA genes in tumors. Subsequently, numerous evidences have been found that point to a role for miRNAs in the pathogenesis of cancer by targeting oncogenes and tumor suppressors. The aim of this project is to elucidate the biological role of miRNA-34c in prostate cancer.
1
Material & Methods: We examined expression of miR-15a in the specimen of prostate needle biopsy and PCa cell lines. We applied real-time PCR method by using stem-loop TaqMan miRNA assay. PCa cells (LNCaP, C4-2, PC3 and DU145) were transfected with has-pre-miRNAs or control RNA. Cell proliferation was determined by XTT assay after 72 hours of treatment. In apoptosis assay, PCa cells were collected and stained with Annexin V and PI, and the percentage of apoptotic cells was analyzed by flow cytometry after 48 hrs of treatment. Cell invasion assay was determined by measuring the widths of lines which have drowned on the cells plated after miR-15a transfection. To identify target genes of miR-15a, we combined genome-wide gene expression analysis and bioinformatics data. Results: The expression levels of miR-15a were significantly reduced in PCa specimens and PCa cells compared to the normal prostate tissue. To characterize the molecular basis of miR-15a tumor suppression in PCa, we investigated the effect of miR-15a on cell proliferation, apoptotic cell induction and cell migration. Transfection of miR-15a caused growth inhibition in LNCaP, C4-2 and PC3 but not in DU145 cells. Apoptotic fraction was significantly increased in LNCaP and C4-2 but not in PC3 and DU145 cells. Invasion assay showed decreased migration ability in PC3 cells but no remarkable change in DU145 cells. Combined experimental results of gene expression analysis and bioinformatics data suggested that several oncogenes including BCL2, and CCND1, were candidate target genes of miR-15a. The mRNA expression levels of BCL2 were decreased in LNCaP and C4-2 but not in PC3 and DU145 by miR-15a transfection. The mRNA of CCND1 was repressed in all the cell lines. Our data suggest that the growth inhibition and induction of apoptosis in LNCaP and C4-2 was BCL2 and CCND1 dependent, and inhibition of cell migration in PC3 might because of suppression of CCND1. DU145 cell might have different tumorigenesis pathways independent from BCL2 and CCND1. Conclusions: miR-15a can inhibit cell proliferation and induce apoptotic cells in hormonedependent and -independent PCa cells by targeting several oncogenes including BCL2 and CCND1. This feature has considerable therapeutic implications for novel treatment of PCa in future.
779
Microarray analysis of miRNA expression in different prostate cancer cell lines Kamradt J.1, Holzmann K.H.2, Tolosi L.3, Wullich B.4, Stöckle M.5, Jung V.5 University of Saarland, Dept. of Urology and Pediatric Urology, Homburg/Saar, Germany, 2University of Ulm, Izkf, Ulm, Germany, 3Max-Planck Institut Für Informatik, Bioinformatik und Angewandte Algorithmik, Saarbrücken, Germany, 4University of Erlangen, Dept. of Urology, Erlangen, Germany, 5University of Saarland, Dept. of Urology and Pediatric Urology, Homburg/Saar, Germany 1
Introduction & Objectives: MicroRNAs (miRNAs) are small non-coding RNA molecules that represent a novel way of modulating gene expression on a posttranscription level. About 3% of human genes encode for miRNAs, and up to 30% of human protein coding genes may be regulated by miRNAs. miRNAs were shown to be important in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. Therefore altered miRNA expression has been implicated to contribute to human disease, including cancer. In the present study we analyzed miRNA expression profiles of benign and several malign prostate cell lines including derived sublines. Material & Methods: MiRNA was isolated from cell lines PC3, PC3-N, PC3-125L, LNCaP, LNCaP-ABL, DU145, DU145MN1, CWR22, CWR22-Rv1 and BPH-1. Cell line miRNA was labeled and hybridized against fibroblast miRNA on a microarray containing 386 different miRNAs in quadriplicates (Ambion mirVana probe set V1). Scanned array images were analyzed with DeArray software, data were further processed and visualized using Bioconductor packages in the statistical program R. Results: Hierarchical clustering of the experimental results showed a clear separation of BPH-1 from all prostate cancer cell lines. Surprisingly, sublines of the different prostate cancer cell lines did not cluster close together as expected. Comparing parental cell lines with their sublines we found a downregulation of miRNA 339 in sublines PC3-N, PC3-125L and DU145-MN1 and an upregulation of miRNA 7051_1 in PC3-N and DU145-MN1. Conclusions: miRNA expression profile does allow distinction between benign and malign prostate cell lines, but also parental cell lines and their sublines have a differential miRNA expression pattern. Interestingly, sublines generated from metastases in animal models, showed a few miRNA changes in common that may indicate to an important function of theses miRNAs in tumor progression.
Material & Methods: A miRNA expression array (Exiqon) was performed to detect levels of miRNA in cancer compared to benign prostatic tissue. The biological effect of miR-34c was studied in the prostate cell lines PC3, DU145, PNT2 and LNCaP by gain-of-function using mimic oligonucleotides (Dharmacon). The effect on cell number (SRB), proliferation (BrdU incorporation), apoptosis (Annexin-V staining), migration (wound healing) and invasion (Matrigel Invasion Chamber) were assessed. Results: We show by miRNA expression array that the expression of miRNA34c is lower in prostate cancer tissue compared to benign prostate tissue (p < 0.0001). Over-expressing miR-34c by mimic oligonucleotides had negative effects on cell number in PC3 (58% decrease after 120h, p < 0.001). The same could be seen in the DU145, LNCaP, and PNT2. The decrease in cell number was due to effects both on cell proliferation (70% decrease) and apoptosis (384% increase p < 0.0001). Furthermore, migration (629% decrease in DU145, p < 0.0001) and invasion (66% decrease in PC3, p < 0.01) was inhibited when over-expressing miR34. We found that over-expressing miR-34c had effects on the protein level of e.g. E2F3 and BCL2 and this is in agreement with the biological effects observed. Conclusions: Our results suggest that a decreased expression of miR-34c in prostate cancer could be one of the contributors to prostate cancer progression.
780
Down-regulation of microRNA-221 hallmarks prostate cancer metastasis and is a predictor of clinical recurrence Spahn M.1, Kneitz S.2, Scholz C.J.2, Stenger N.1, Rüdiger T.3, Ströbel P.4, Riedmiller H.1, Kneitz B.1 University Medical School, University of Würzburg, Dept. of Urology and Pediatric Urology, Würzburg, Germany, 2University of Würzburg, Micro-Array Core Unite, Izkf (Interdisciplinary Center For Clinical Research), Würzburg, Germany, 3Community Hospital Karlsruhe, Institute of Pathology, Karlsruhe, Germany, 4University Medical Center Mannheim, Institute of Pathology, Mannheim, Germany 1
Introduction & Objectives: Emerging evidence shows that microRNAs (miR) are involved in the pathogenesis of a variety of cancers, including prostate carcinoma. Little information is available regarding miR expression levels in lymph node metastasis of prostate cancer or the potential of miRs as prognostic markers in this disease. Therefore, we analyzed miR signatures in prostate carcinoma metastasis and studied the role of miR-221 as a novel prognostic marker in prostate cancer. Material & Methods: We analysed the global expression of miRs in benign and hyperplastic prostate tissue (BPH), primary prostate carcinoma (PCA), and corresponding metastatic tissues by micro-array analysis. Ninety two samples of radical prostatectomies were subsequently investigated by qRT-PCR to validate the associations between the expression of miR-221, various clinicopathologic factors, and patient survival. Results: Consistent with the proposal that some microRNAs are oncomirs, we found aberrant expression of several miRs, including the down-regulation of miR221, in prostate carcinoma metastasis. In a large study cohort, the miR-221 oncomir was progressively down-regulated in aggressive forms of prostate carcinoma. Down-regulation of miR-221 was associated with clinicopathological parameters, including the Gleason score and the clinical recurrence during follow up. Kaplan Meier estimates and Cox proportional hazard models showed that miR-221 downregulation was linked to tumor progression and recurrence. Conclusions: Our results suggest that progressive miR-221 down-regulation is a hallmark of metastasis and a novel prognostic marker in prostate carcinoma. This suggests that miR-221 has potential as a diagnostic marker and therapeutic target.
Eur Urol Suppl 2009;8(4):315
777
778
miR-15a functions as a tumor suppressor for prostate cancer by targeting multiple oncogenes
Upregulation of microRNA-34c affect proliferation, migration, invasion and apoptosis in prostate cells
Kojima S.1, Watanabe R.1, Tsukada J.2, Furuya Y.1, Seki N.2
Hagman Z.H., Ceder A.Y.C.
Teikyo University Chiba Medical Center, Dept. of Urology, Ichihara, Japan, 2Chiba University School of Medicine, Dept. of Functional Genomics, Chiba, Japan
Clinical Chemistry, Dept. of Laboratory Medicine, Malmö, Sweden
Introduction & Objectives: MicroRNAs (miRNAs) are short noncoding RNAs regulating gene expression that play roles in human cancer. Each miRNA is predicted to regulate hundreds of transcripts, but few have been experimentally validated. The miR-15a cluster resides at chromosome 13q14.3, a genomic region frequently deleted in human cancers including prostate cancer (PCa). Our previous works indicated that expression levels of miR-15a were down-regulated in PCa specimens. Here, to investigate the role of tumorigenesis of miR-15a as tumor suppressors in PCa. In addition, we subjected to target gene prediction of miR-15a using genome-wide gene expression analysis.
Introduction & Objectives: MicroRNA (miRNA) are naturally occurring short noncoding RNA (18-25 nt) that regulate gene expression by inhibiting translation of their target mRNA by non perfect base pairing to the 3’UTRs. To date, nearly 700 human miRNAs have been identified, but this is expected to increase to around 1000. Recently, miRNAs was suggested to be of relevance for cancer progression after discovering that the miRNA expression pattern change and that there are recurrent amplification and deletion of miRNA genes in tumors. Subsequently, numerous evidences have been found that point to a role for miRNAs in the pathogenesis of cancer by targeting oncogenes and tumor suppressors. The aim of this project is to elucidate the biological role of miRNA-34c in prostate cancer.
1
Material & Methods: We examined expression of miR-15a in the specimen of prostate needle biopsy and PCa cell lines. We applied real-time PCR method by using stem-loop TaqMan miRNA assay. PCa cells (LNCaP, C4-2, PC3 and DU145) were transfected with has-pre-miRNAs or control RNA. Cell proliferation was determined by XTT assay after 72 hours of treatment. In apoptosis assay, PCa cells were collected and stained with Annexin V and PI, and the percentage of apoptotic cells was analyzed by flow cytometry after 48 hrs of treatment. Cell invasion assay was determined by measuring the widths of lines which have drowned on the cells plated after miR-15a transfection. To identify target genes of miR-15a, we combined genome-wide gene expression analysis and bioinformatics data. Results: The expression levels of miR-15a were significantly reduced in PCa specimens and PCa cells compared to the normal prostate tissue. To characterize the molecular basis of miR-15a tumor suppression in PCa, we investigated the effect of miR-15a on cell proliferation, apoptotic cell induction and cell migration. Transfection of miR-15a caused growth inhibition in LNCaP, C4-2 and PC3 but not in DU145 cells. Apoptotic fraction was significantly increased in LNCaP and C4-2 but not in PC3 and DU145 cells. Invasion assay showed decreased migration ability in PC3 cells but no remarkable change in DU145 cells. Combined experimental results of gene expression analysis and bioinformatics data suggested that several oncogenes including BCL2, and CCND1, were candidate target genes of miR-15a. The mRNA expression levels of BCL2 were decreased in LNCaP and C4-2 but not in PC3 and DU145 by miR-15a transfection. The mRNA of CCND1 was repressed in all the cell lines. Our data suggest that the growth inhibition and induction of apoptosis in LNCaP and C4-2 was BCL2 and CCND1 dependent, and inhibition of cell migration in PC3 might because of suppression of CCND1. DU145 cell might have different tumorigenesis pathways independent from BCL2 and CCND1. Conclusions: miR-15a can inhibit cell proliferation and induce apoptotic cells in hormonedependent and -independent PCa cells by targeting several oncogenes including BCL2 and CCND1. This feature has considerable therapeutic implications for novel treatment of PCa in future.
779
Microarray analysis of miRNA expression in different prostate cancer cell lines Kamradt J.1, Holzmann K.H.2, Tolosi L.3, Wullich B.4, Stöckle M.5, Jung V.5 University of Saarland, Dept. of Urology and Pediatric Urology, Homburg/Saar, Germany, 2University of Ulm, Izkf, Ulm, Germany, 3Max-Planck Institut Für Informatik, Bioinformatik und Angewandte Algorithmik, Saarbrücken, Germany, 4University of Erlangen, Dept. of Urology, Erlangen, Germany, 5University of Saarland, Dept. of Urology and Pediatric Urology, Homburg/Saar, Germany 1
Introduction & Objectives: MicroRNAs (miRNAs) are small non-coding RNA molecules that represent a novel way of modulating gene expression on a posttranscription level. About 3% of human genes encode for miRNAs, and up to 30% of human protein coding genes may be regulated by miRNAs. miRNAs were shown to be important in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. Therefore altered miRNA expression has been implicated to contribute to human disease, including cancer. In the present study we analyzed miRNA expression profiles of benign and several malign prostate cell lines including derived sublines. Material & Methods: MiRNA was isolated from cell lines PC3, PC3-N, PC3-125L, LNCaP, LNCaP-ABL, DU145, DU145MN1, CWR22, CWR22-Rv1 and BPH-1. Cell line miRNA was labeled and hybridized against fibroblast miRNA on a microarray containing 386 different miRNAs in quadriplicates (Ambion mirVana probe set V1). Scanned array images were analyzed with DeArray software, data were further processed and visualized using Bioconductor packages in the statistical program R. Results: Hierarchical clustering of the experimental results showed a clear separation of BPH-1 from all prostate cancer cell lines. Surprisingly, sublines of the different prostate cancer cell lines did not cluster close together as expected. Comparing parental cell lines with their sublines we found a downregulation of miRNA 339 in sublines PC3-N, PC3-125L and DU145-MN1 and an upregulation of miRNA 7051_1 in PC3-N and DU145-MN1. Conclusions: miRNA expression profile does allow distinction between benign and malign prostate cell lines, but also parental cell lines and their sublines have a differential miRNA expression pattern. Interestingly, sublines generated from metastases in animal models, showed a few miRNA changes in common that may indicate to an important function of theses miRNAs in tumor progression.
Material & Methods: A miRNA expression array (Exiqon) was performed to detect levels of miRNA in cancer compared to benign prostatic tissue. The biological effect of miR-34c was studied in the prostate cell lines PC3, DU145, PNT2 and LNCaP by gain-of-function using mimic oligonucleotides (Dharmacon). The effect on cell number (SRB), proliferation (BrdU incorporation), apoptosis (Annexin-V staining), migration (wound healing) and invasion (Matrigel Invasion Chamber) were assessed. Results: We show by miRNA expression array that the expression of miRNA34c is lower in prostate cancer tissue compared to benign prostate tissue (p < 0.0001). Over-expressing miR-34c by mimic oligonucleotides had negative effects on cell number in PC3 (58% decrease after 120h, p < 0.001). The same could be seen in the DU145, LNCaP, and PNT2. The decrease in cell number was due to effects both on cell proliferation (70% decrease) and apoptosis (384% increase p < 0.0001). Furthermore, migration (629% decrease in DU145, p < 0.0001) and invasion (66% decrease in PC3, p < 0.01) was inhibited when over-expressing miR34. We found that over-expressing miR-34c had effects on the protein level of e.g. E2F3 and BCL2 and this is in agreement with the biological effects observed. Conclusions: Our results suggest that a decreased expression of miR-34c in prostate cancer could be one of the contributors to prostate cancer progression.
780
Down-regulation of microRNA-221 hallmarks prostate cancer metastasis and is a predictor of clinical recurrence Spahn M.1, Kneitz S.2, Scholz C.J.2, Stenger N.1, Rüdiger T.3, Ströbel P.4, Riedmiller H.1, Kneitz B.1 University Medical School, University of Würzburg, Dept. of Urology and Pediatric Urology, Würzburg, Germany, 2University of Würzburg, Micro-Array Core Unite, Izkf (Interdisciplinary Center For Clinical Research), Würzburg, Germany, 3Community Hospital Karlsruhe, Institute of Pathology, Karlsruhe, Germany, 4University Medical Center Mannheim, Institute of Pathology, Mannheim, Germany 1
Introduction & Objectives: Emerging evidence shows that microRNAs (miR) are involved in the pathogenesis of a variety of cancers, including prostate carcinoma. Little information is available regarding miR expression levels in lymph node metastasis of prostate cancer or the potential of miRs as prognostic markers in this disease. Therefore, we analyzed miR signatures in prostate carcinoma metastasis and studied the role of miR-221 as a novel prognostic marker in prostate cancer. Material & Methods: We analysed the global expression of miRs in benign and hyperplastic prostate tissue (BPH), primary prostate carcinoma (PCA), and corresponding metastatic tissues by micro-array analysis. Ninety two samples of radical prostatectomies were subsequently investigated by qRT-PCR to validate the associations between the expression of miR-221, various clinicopathologic factors, and patient survival. Results: Consistent with the proposal that some microRNAs are oncomirs, we found aberrant expression of several miRs, including the down-regulation of miR221, in prostate carcinoma metastasis. In a large study cohort, the miR-221 oncomir was progressively down-regulated in aggressive forms of prostate carcinoma. Down-regulation of miR-221 was associated with clinicopathological parameters, including the Gleason score and the clinical recurrence during follow up. Kaplan Meier estimates and Cox proportional hazard models showed that miR-221 downregulation was linked to tumor progression and recurrence. Conclusions: Our results suggest that progressive miR-221 down-regulation is a hallmark of metastasis and a novel prognostic marker in prostate carcinoma. This suggests that miR-221 has potential as a diagnostic marker and therapeutic target.
Eur Urol Suppl 2009;8(4):315