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PDF file - 25K, List of sequences of cloning primers, mutagenesis primers, and Northern blotting probes.

PDF file - 85K, Results of Illumina sequencing of small RNAs with corresponding relative expression of miRNAs. The miRNA expression in five RNA pools of normal, normal prostate, pT2, pT3 and lymph node metastasizing (pN1) PCa was analyzed by deep sequencing. The absolute number of all identified transcripts in the five cDNA libraries, as well as relative expression as a percentage of the total miRNA count, is given for every miRNA found.

PDF file - 26K, Clinicopathological characteristics of prostate carcinoma samples used for Illumina sequencing and qRT-PCR validation of the sequencing. The clinicopathological characteristics of the patient set used for deep sequencing are given in the left column. The properties of the sample set used for qRT-PCR are summarized on the right side.

Supplementary Data from The MicroRNA Profile of Prostate Carcinoma Obtained by Deep Sequencing

PDF file - 96K, Control experiments carried out in parallel. The individual control experiments shown in Figure 5A and 5B were carried out in parallel to show that the changes in growth behavior were not due to different cell culture conditions. We consistently observe a cytotoxic effect of the empty pSG5 vector on cell growth at the 72h time point.

PDF file - 123K, Analysis of miR-15a and miR-200b expression by Northern blotting (A) Ectopic expression of miR-15a and miR-200b. 293T cells were transfected either with pSG5 vector or with the pSG5-miR-15a or-200b expression plasmids. The ectopic expression of the miRNAs was detected by Northern blotting using specific radioactive labeled probes. (B) Endogenous expression of miR-15a and miR-200b in PCa cell lines DU145 and LNCaP. Total RNA was extracted from the cell lines PNF, DU145 and LNCaP and analyzed by Northern blotting using specific radioactive labeled probes against miR-15a and -200b. tRNA served as loading control.

PDF file - 205K, Validation of the microRNA expression profile by Northern blotting. For confirmation of the results obtained by the sequencing the relative expression levels of miR-375, -148a, -200b, -20a, -15a, -145, -143 and let-7c in the RNA pools used for sequencing were analyzed by northern blotting using specific radiolabeled probes. tRNA served as loading control. A PhosphoImager? was used for visualization of the hybridized probes.

Supplementary Figures 1-6 from Downregulation of Sec23A Protein by miRNA-375 in Prostate Carcinoma

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529–38. ©2010 AACR.

PDF file - 175K, The relative expression of SEC23A, PHLPP1 and PHLPP2 determined by qRT-PCR. (A-C) The relative expression of SEC23A, PHLPP1, and PHLPP2 mRNA was analyzed by qRT-PCR in a patient set of 40 pairs of normal and corresponding pT2 (N0), pT3 (N0) and lymph node metastasizing (pN1) PCa tissues.

PDF file - 175K, qRT-PCR analysis of miRNAs found deregulated by sequencing. 40 paired samples of prostate carcinoma and the corresponding non-tumor tissues were analyzed by qRT-PCR. In the overall comparison, the changes in miR-20a, -143, -145, -148a, -200b and -375 were statistically significant. MiR-15a and let7c revealed no significant change.

Prostate carcinoma (CaP) is a leading cause of cancer-related death in men. We have previously determined the microRNA (miRNA) profile of primary CaP in comparison with nontumor prostate tissue. miRNAs are small, noncoding RNAs that inhibit protein synthesis on a posttranscriptional level by binding to the 3′-untranslated region (3′-UTR) of their target genes. In primary CaP tissue, we have previously found by miRNA sequencing that miR-375 and miR-200c were upregulated 9.1- and 4.5-fold, respectively. A computational analysis predicted the 3′-UTR of the SEC23A gene as a potential target for both miR-375 and miR-200c. Here, we show that the 3′-UTR of SEC23A mRNA is indeed a target for miR-375 and miR-200c and that both miRNAs downregulate Sec23A protein expression when ectopically expressed in human 293T cells. In primary samples of CaP, we found a direct correlation between reduction of SEC23A mRNA and overexpression of miR-375 but not of miR-200c. The reduced levels of Sec23A protein were inversely correlated to the increased amount of miR-375 in the LNCaP and DU145 CaP cell lines when compared with normal prostate fibroblasts. In primary CaP, we also detected decreased amounts of Sec23A protein when compared with corresponding normal prostate tissue. Ectopically overexpressed Sec23A in LNCaP and DU145 CaP cells significantly reduced the growth properties, indicating that Sec23A might play a role in the induction or growth of prostate carcinoma. Sec23A overexpression reduced cell growth but did not induce apoptosis, whereas inhibition of Sec23A stimulated cell proliferation. Mol Cancer Res; 9(6); 791–800. ©2011 AACR.

MicroRNAs (miRNA) posttranscriptionally regulate gene expression and are important in tumorigenesis. Previous deep sequencing identified the miRNA profile of prostate carcinoma versus nonmalignant prostate tissue. Here, we generated miRNA expression profiles of prostate carcinoma by deep sequencing, with increasing tumor stage relative to corresponding nonmalignant and healthy prostate tissue, and detected clearly changed miRNA expression patterns. The miRNA profiles of the healthy and nonmalignant tissues were consistent with our previous findings, indicating a high fidelity of the method employed. In the tumors, quantitative real-time PCR (qRT-PCR) analysis of 40 paired samples of prostate carcinoma versus normal tissue revealed significant upregulation of miR-20a, miR-148a, miR-200b, and miR-375 and downregulation of miR-143 and miR-145. Hereby, miR-375 increased from normal to organ-confined tumors (pT2 pN0), slightly decreased in tumors with extracapsular growth (pT3 pN0), but was then expressed again at higher levels in lymph node metastasizing (pN1) tumors. The sequencing data for miR-375 were confirmed by Northern blotting and qRT-PCR. The regulation for other selected miRNAs could, however, not be confirmed by qRT-PCR in individual tumor stages. MiR-200b, in addition to miR-200c and miR-375 reduced the expression of SEC23A. Interestingly, miR-375, found by sequencing in pT2 upregulated by us and others in tumor versus normal tissue, and miR-15a, found by sequencing in pT2 and pT3 and in the metastasizing tumors, target the phosphatases PHLPP1 and PHLPP2, respectively. PHLPP1 and PHLPP2 dephosphorylate members of the AKT family of signal transducers, thereby inhibiting cell growth. Coexpression of miR-15a and miR-375 resulted in downregulation of PHLPP1/2 and strongly increased prostate carcinoma cell growth.Implications: These genomic data reveal relevant miRNAs in prostate cancer that may have biomarker and therapeutic potential. Mol Cancer Res; 12(2); 250–63. ©2013 AACR.

The histogenesis of prostatic neuroendocrine cells is controversial: a stem cell hypothesis with a urogenital sinus-derived progeny of all prostatic epithelial cells is opposed by a dual origin hypothesis, favoring the derivation of neuroendocrine cells from the neural crest, with the secretory and basal cells being of urogenital sinus origin. A computer-assisted 3D reconstruction was used to analyze the distribution of chromogranin A immunoreactive cells in serial sections of human fetal prostate specimens (gestation weeks 18 and 25). Immunohistochemical double labeling studies with YFP and serotonin antisera combined with electron microscopy were carried out on double-transgenic Wnt1-Cre/ROSA26-YFP mice showing stable YFP expression in all neural crest-derived cell populations despite loss of Wnt1 expression. 3D reconstruction of the distribution pattern of neuroendocrine cells in the human fetal prostate indicates a migration of paraganglionic cells passing the stroma and reaching the prostate ducts. Double-transgenic mice showed 55% double labeling of periurethral neuroendocrine cells expressing both serotonin and YFP, whereas single serotonin labeling was observed in 36% and exclusive YFP labeling in 9%. The results favor the assumption of a major fraction of neural crest-derived neuroendocrine cells in both the human and murine prostates. OA hybrid

Prostate cancer is a leading cause of cancer mortality in men. One of the distinct characteristics of prostate cancer is over-expression of the ERG proto-oncogene. The TMPRSS2-ERG gene fusion, the most common gene fusion, is found in approximately 50% of prostate cancer cases. We show that certain microRNAs are extensively deregulated in prostate cancer cell lines and primary clinical cancer samples. MicroRNAs are capable of modulating post-transcriptional gene expression via inhibition of protein synthesis. Independent target prediction methods have indicated that the 3' untranslated region of the ERG mRNA is a potential target of miR-145. miR-145 is consistently down-regulated in prostate cancer. Here we show that the ERG 3' untranslated region is a regulative target of miR-145 in vitro. Ectopic expression of miR-145 led to a reduction in expression of the ERG protein. We analyzed 26 prostate cancer samples and corresponding normal tissue. ERG protein expression was found to be elevated in the tumor samples, together with increased expression of several ERG isoforms. We identified ERG proteins of 35 and 24 kDa, which may represent unknown ERG splice variants. Analyses of miR-145 and ERG mRNA expression revealed a general down-regulation of miR-145 irrespective of the presence or absence of translocations involving ERG. This observation indicates that down-regulation of miR-145 may contribute to the increased expression of most ERG splice variants sharing the miR-145 target sequence in their 3' untranslated region.

Posttranscriptional gene regulation by microRNAs (miRNAs) contributes to the induction and maintenance of prostate carcinoma (PCa). To identify mRNAs enriched or removed from Ago2-containing RISC complexes, these complexes were immunoprecipitated from normal prostate fibroblasts (PNFs) and the PCa line DU145 and the bound mRNAs were quantified by microarray. The analysis of Ago complexes derived from PNFs or DU145 confirmed the enrichment or depletion of a variety of mRNAs already known from the literature to be deregulated. Novel potential targets were analyzed by luciferase assays with miRNAs known to be deregulated in PCa. We demonstrate that the mRNAs of the death effector domain-containing protein (DEDD), the tumor necrosis factor receptor superfamily, member 10b protein (TNFRSF10B), the tumor protein p53 inducible nuclear protein 1 (TP53INP1), and the secreted protein, acidic, cysteine-rich (SPARC; osteonectin) are regulated by miRNAs miR-148a, miR-20a, miR-24, and miR-29a/b, respectively. Therefore, these miRNAs represent potential targets for therapy. Surprisingly, overexpression of miR-24 induced focus formation and proliferation of DU145 cells, while miR-29b reduced proliferation. The study confirms genes deregulated in PCa by virtue of their presence/absence in the Ago2-complex. In conjunction with the already published miRNA profiles of PCa, the data can be used to identify miRNA-regulated mRNAs.

Prostate carcinoma (CaP) is a leading cause of cancer-related death in men. We have previously determined the microRNA (miRNA) profile of primary CaP in comparison with nontumor prostate tissue. miRNAs are small, noncoding RNAs that inhibit protein synthesis on a posttranscriptional level by binding to the 3′-untranslated region (3′-UTR) of their target genes. In primary CaP tissue, we have previously found by miRNA sequencing that miR-375 and miR-200c were upregulated 9.1- and 4.5-fold, respectively. A computational analysis predicted the 3′-UTR of the SEC23A gene as a potential target for both miR-375 and miR-200c. Here, we show that the 3′-UTR of SEC23A mRNA is indeed a target for miR-375 and miR-200c and that both miRNAs downregulate Sec23A protein expression when ectopically expressed in human 293T cells. In primary samples of CaP, we found a direct correlation between reduction of SEC23A mRNA and overexpression of miR-375 but not of miR-200c. The reduced levels of Sec23A protein were inversely correlated to the increased amount of miR-375 in the LNCaP and DU145 CaP cell lines when compared with normal prostate fibroblasts. In primary CaP, we also detected decreased amounts of Sec23A protein when compared with corresponding normal prostate tissue. Ectopically overexpressed Sec23A in LNCaP and DU145 CaP cells significantly reduced the growth properties, indicating that Sec23A might play a role in the induction or growth of prostate carcinoma. Sec23A overexpression reduced cell growth but did not induce apoptosis, whereas inhibition of Sec23A stimulated cell proliferation. Mol Cancer Res; 9(6); 791–800. ©2011 AACR.

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression via posttranscriptional inhibition of protein synthesis. They play a vital role in tumorigenesis. To characterize the diagnostic potential of miRNAs in prostate cancer, a leading cause of cancer mortality, we performed screening of miRNA expression profiles. We used commercially available microarrays to establish miRNA expression profiles from a cohort of 20 cancer samples. The expression of selected miRNAs was analyzed by quantitative real-time PCR and the identity of miRNA expressing cells was determined by miRNA in situ hybridization. We identified 25 miRNAs that showed a significant differential expression in cancer samples. The comparison with previously published data generated by deep sequencing of cDNA libraries of small RNA molecules revealed a concordance rate of 47% among miRNAs identified with both techniques. The differential expression of miRNAs miR-375, miR-143 and miR-145 was validated by quantitative PCR. MiRNA in situ hybridization revealed that the differential expression is cancer-cell associated. A combination of three miRNAs correctly classified tissue samples with an accuracy of 77.6% with an area under the receiver–operator characteristic curve of 0.810. Our data extend the knowledge about the deregulation of miRNAs in prostate cancer. The differential expression of several miRNAs is highly consistent using independent cohorts of tumor samples, different tissue preservation methods and different experimental methods. Our results indicate that combinations of miRNAs are promising biomarkers for the diagnosis of prostate cancer. MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression via posttranscriptional inhibition of protein synthesis. They play a vital role in tumorigenesis. To characterize the diagnostic potential of miRNAs in prostate cancer, a leading cause of cancer mortality, we performed screening of miRNA expression profiles. We used commercially available microarrays to establish miRNA expression profiles from a cohort of 20 cancer samples. The expression of selected miRNAs was analyzed by real-time quantitative PCR (qPCR) and the identity of miRNA expressing cells was determined by miRNA in situ hybridization. We identified 25 miRNAs that showed a significant differential expression in cancer samples. The comparison with previously published data generated by deep sequencing of cDNA libraries of small RNA molecules revealed a concordance rate of 47% among miRNAs identified with both techniques. The differential expression of miRNAs miR-375, miR-143 and miR-145 was validated by qPCR. MiRNA in situ hybridization revealed that the differential expression is cancer-cell associated. A combination of three miRNAs correctly classified tissue samples with an accuracy of 77.6% with an area under the ROC curve of 0.810. Our data extend the knowledge about the deregulation of miRNAs in prostate cancer. The differential expression of several miRNAs is highly consistent using independent cohorts of tumor samples, different tissue preservation methods and different experimental methods. Our results indicate that combinations of miRNAs are promising biomarkers for the diagnosis of prostate cancer.

Prostate cancer is a leading cause of tumor mortality. To characterize the underlying molecular mechanisms, we have compared the microRNA (miRNA) profile of primary prostate cancers and noncancer prostate tissues using deep sequencing. MiRNAs are small noncoding RNAs of 21 to 25 nucleotides that regulate gene expression through the inhibition of protein synthesis. We find that 33 miRNAs were upregulated or downregulated >1.5-fold. The deregulation of selected miRNAs was confirmed by both Northern blotting and quantitative reverse transcription-PCR in established prostate cancer cell lines and clinical tissue samples. A computational search indicated the 3′-untranslated region (UTR) of the mRNA for myosin VI (MYO6) as a potential target for both miR-143 and miR-145, the expression of which was reduced in the tumor tissues. Upregulation of myosin VI in prostate cancer was previously shown by immunohistochemistry. The level of MYO6 mRNA was significantly induced in all primary tumor tissues compared with the nontumor tissue from the same patient. This finding was matched to the upregulation of myosin VI in established prostate cancer cell lines. In luciferase reporter analysis, we find a significant negative regulatory effect on the MYO6 3′UTR by both miR-143 and miR-145. Mutation of the potential binding sites for miR-143 and miR-145 in the MYO6 3′UTR resulted in a loss of responsiveness to the corresponding miRNA. Our data indicate that miR-143 and miR-145 are involved in the regulation of MYO6 expression and possibly in the development of prostate cancer. Mol Cancer Res; 8(4); 529–38. ©2010 AACR.

MicroRNAs (miRNA) posttranscriptionally regulate gene expression and are important in tumorigenesis. Previous deep sequencing identified the miRNA profile of prostate carcinoma versus nonmalignant prostate tissue. Here, we generated miRNA expression profiles of prostate carcinoma by deep sequencing, with increasing tumor stage relative to corresponding nonmalignant and healthy prostate tissue, and detected clearly changed miRNA expression patterns. The miRNA profiles of the healthy and nonmalignant tissues were consistent with our previous findings, indicating a high fidelity of the method employed. In the tumors, quantitative real-time PCR (qRT-PCR) analysis of 40 paired samples of prostate carcinoma versus normal tissue revealed significant upregulation of miR-20a, miR-148a, miR-200b, and miR-375 and downregulation of miR-143 and miR-145. Hereby, miR-375 increased from normal to organ-confined tumors (pT2 pN0), slightly decreased in tumors with extracapsular growth (pT3 pN0), but was then expressed again at higher levels in lymph node metastasizing (pN1) tumors. The sequencing data for miR-375 were confirmed by Northern blotting and qRT-PCR. The regulation for other selected miRNAs could, however, not be confirmed by qRT-PCR in individual tumor stages. MiR-200b, in addition to miR-200c and miR-375 reduced the expression of SEC23A. Interestingly, miR-375, found by sequencing in pT2 upregulated by us and others in tumor versus normal tissue, and miR-15a, found by sequencing in pT2 and pT3 and in the metastasizing tumors, target the phosphatases PHLPP1 and PHLPP2, respectively. PHLPP1 and PHLPP2 dephosphorylate members of the AKT family of signal transducers, thereby inhibiting cell growth. Coexpression of miR-15a and miR-375 resulted in downregulation of PHLPP1/2 and strongly increased prostate carcinoma cell growth. Implications: These genomic data reveal relevant miRNAs in prostate cancer that may have biomarker and therapeutic potential. Mol Cancer Res; 12(2); 250–63. ©2013 AACR.

In primary prostate cancer (PCa), a major cause of cancer-related death in men, the expression of various microRNAs (miRNAs) is deregulated. We previously detected several miRNAs, for example, miR-24 and miR-22, as significantly downregulated in PCa (Szczyrba et al., Mol Cancer Res 2010;8:529-38). An in silico search predicted that zinc finger protein 217 (ZNF217) and importin 7 (IPO7) were potential target genes of these miRNAs. Additionally, for two genes that are deregulated in PCa (heterogeneous nuclear ribonucleoprotein K, hnRNP-K, and vascular endothelial growth factor A, VEGF-A), we identified two regulatory miRNAs, miR-205 and miR-29b. The regulation of the 3'-untranslated regions of the four genes by their respective miRNAs was confirmed by luciferase assays. As expected, the upregulation of ZNF217, hnRNP-K, VEGF-A and IPO7 could be verified at the protein level in the PCa cell lines LNCaP and DU145. ZNF217 and IPO7, which had not yet been studied in PCa, were analyzed in more detail. ZNF217 mRNA is overexpressed in primary PCa samples, and this overexpression translates to an elevated protein level. However, IPO7 was upregulated at the protein level alone. The inhibition of ZNF217 and IPO7 by siRNA resulted in reduced proliferation of the PCa cell lines. ZNF217 could thus be identified as an oncogene that is overexpressed in PCa and affects the growth of PCa cell lines, whereas the function of IPO7 remains to be elucidated in greater detail.

Background: MicroRNAs (miRNAs) are short, non-coding RNAs that post-transcriptionally regulate gene expression and play an important role in tumorigenesis. Previous deep-sequencing identified the microRNA profile of prostate carcinoma (PCa) vs. non-malignant prostate tissue. As an extension of our recent analyses, we now compared organ-confined prostate tumors (pT2), tumors with extra capsular growth (pT3, pN0), and lymph node metastasizing (pN1) PCa relative to corresponding non-malignant prostate tissue and healthy prostate tissue. Material and Methods: We performed comparative miRNA expression profiling using deep sequencing of cDNA libraries. Potential target genes of deregulated miRNAs were identified by database analysis and subsequently validated in vitro using reporter gene constructs. miRNA and mRNA expression was determined using quantitative real-time PCR. Results: The miRNA expression from the healthy and non-malignant tissue is consistent with our previous findings, indicating a high fidelity of the method employed. Some miRNAs (miR-375, miR-148a, miR-141 and miR-21) are expressed at increasing levels with increasing tumor stage, while other miRNAs (miR-200b/c, miR-25, miR-17 or miR-93) are up-regulated only from organ-confined tumors (pT2) to tumors with extra-capsular growth (pT3) but then expressed at lower levels after the transition to lymph node metastasizing tumors (pN0 vs. pN1). The same trend was seen in qRT-PCR analysis but due to the limited sample number, no significance was reached. Interestingly, miR-375, found to be up-regulated in tumor vs. normal tissue, and miR-15a, found induced by sequencing in T2 and T3 tumors but not in lymph node metastasizing tumors, target the phosphatases PHLPP1 and PHLPP2, respectively. PHLPP1 and PHLPP2 dephosphorylate members of the AKT-family of signal transducers thereby inhibiting cell growth. Co-expression of miR-15a and miR-375 resulted in down-regulation of PHLPP1/2 and a strongly increased PCa cell growth. Conclusion: miRNA profiling of pT2 (pN0), pT3 (pN0), and lymph node metastasizing prostate carcinoma (pN1) by deep sequencing revealed clearly changed miRNA levels. Citation Format: Elke Nolte, Martin Hart, Sven Wach, Jaroslaw Szczyrba, Helge Taubert, Tilman Rau, Arndt Hartmann, Friedrich A. Grässer, Bernd Wullich. Comparative microRNA profiling of prostate carcinomas with increasing tumor stage by deep-sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4009. doi:10.1158/1538-7445.AM2015-4009

Background: Prostate cancer is one of the leading causes of tumor-related death in men. To identify the underlying mechanisms, microRNA (miRNA) expression profiles of primary prostate cancer samples and of adjacent non-tumor tissue were analyzed. Using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNA miR-24. Material and Methods: We performed comparative miRNA expression profiling using deep sequencing of cDNA libraries. Potential target genes of deregulated miRNAs were identified by database analysis and subsequently validated in vitro using reporter gene constructs. miRNA and mRNA expression was determined using quantitative real-time PCR. Results: One of the miRNAs consistently deregulated in primary prostate cancer samples was miR-24 which was found downregulated in tumor samples more than 1.5-fold in the deep sequencing approach. Independent target prediction databases (including miRanda, Targetscan and PicTar) indicated that the 3’ untranslated region (3’UTR) of the zinc finger protein 217 (ZNF217) gene is a regulative target for miR-24. This prediction could be confirmed using a luciferase reporter gene construct containing the ZNF217 3’UTR. Co-expression of the reporter gene construct with miR-24 resulted in a reduction of reporter gene activity while mutation of the miRNA binding sites caused a loss of responsiveness to the respective miRNA. Furthermore we could show that transient expression of miR-24 reduced the amount of ZNF217 protein in DU145 and LNCaP prostate cancer cells. miR-24 expression was significantly reduced in tumor tissue compared to matched non-tumor tissue (p In Western blot analyses 61% (14/23) of primary tissue samples also displayed an increased amount of ZNF217 protein. A transient knock-down of ZNF217 in DU145 and LNCaP cells applying siRNA technique led to reduced cell proliferation over a time period of 72 hours. Conclusion: Our findings demonstrate that the 3’-UTR of ZNF217 is one regulative target of miR-24, which is commonly deregulated in prostate cancer. ZNF217 mRNA and protein is elevated in the majority of tumor samples analyzed. A transient knock-down of ZNF217 leads to a reduced proliferation in prostate cancer cell lines. Citation Format: Elke Nolte, Jaroslaw Szczyrba, Martin Hart, Celina Döll, Sven Wach, Helge Taubert, Bastian Keck, Elisabeth Kremmer, Robert Stöhr, Arndt Hartmann, Bernd Wullich, Friedrich Grässer. miR-24 influences proliferation of prostate cancer cells in vitro via targeting ZNF217. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3090. doi:10.1158/1538-7445.AM2013-3090

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify the underlying mechanisms we have analyzed microRNA (miRNA) expression profiles of primary prostate cancers and non-cancer prostate tissue. As recently reported by our group, miR-375 was found to be upregulated in prostate cancer using deep sequencing and microarray analyses. We now set out to study whether there is an association between miR-375 expression and the expression of different gene members of the epithelial-mesenchymal transition (EMT) pathway. Material and Methods: Expression of miR-375 and genes of the EMT pathway (ZEB1, ZEB2, N-Cadherin) was measured by qRT-PCR in 26 pairs of prostate cancer and corresponding non-tumor prostate tissue. Bivariate correlation analyses of miR-375 and EMT members as well as for miR-375 and clinicopathological features were carried out. Results: MiR-375 showed a significant upregulation in prostate cancer using qRT-PCR (p=0.0019, paired student's t-test), whereas ZEB1, ZEB2, and N-Cadherin were consistently found downregulated (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3151. doi:1538-7445.AM2012-3151

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify the underlying mechanisms we have analyzed microRNA (miRNA) expression profiles of primary prostate cancers and non-cancer prostate tissue. Using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs and to analyze the physiological consequences of miRNA deregulation using cell culture models. Material and Methods: We performed comparative miRNA expression profiling using deep sequencing of cDNA libraries and microarray analyses. Potential target genes of deregulated miRNAs were identified by database analysis and subsequently validated in vitro using reporter gene constructs. The impact of identified target genes on cell growth was examined by a standardized wound healing assay. Results: Two of the miRNAs consistently deregulated in primary prostate cancer samples were miR-200c and miR-375 which were upregulated more than 1.5-fold in deep sequencing and 2.9-fold and 3.9-fold, respectively, in microarray experiments (p Conclusion: Our findings demonstrate that the 3’-UTR of Sec23a is one regulative target of miR-200c and miR-375, which are commonly deregulated in prostate cancer. Sec23a is reduced in the majority of tumor samples analyzed and has a vital impact on cell growth as determined by wound healing experiments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3961. doi:10.1158/1538-7445.AM2011-3961

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify the underlying mechanisms we have analyzed microRNA (miRNA) expression profiles of primary prostate cancers and non-cancer prostate tissue. Using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs. Material and Methods: We performed comparative miRNA expression profiling using deep sequencing of cDNA libraries and microarray analyses. Potential target genes of deregulated miRNAs were identified by database analysis and subsequently validated in vitro using reporter gene constructs. The abundance of ERG as one of the identified target genes was assessed by Western blotting using a self-generated polyclonal antiserum. Results: One of the miRNAs consistently deregulated in primary prostate cancer samples was miR-145 which was down regulated more than 1.5-fold in deep sequencing and 1.44-fold in microarray experiments (p Conclusion: Our findings demonstrate that the 3’-UTR of ERG is one regulative target of miR-145, which is commonly deregulated in prostate cancer. The ERG gene is commonly overexpressed in prostate cancer and the relative abundance of ERG isoforms is altered in samples of primary prostate cancers. This is the first report to show that ERG is a target of miR-145. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3951. doi:10.1158/1538-7445.AM2011-3951

Background: Prostate cancer is a leading cause of tumor mortality. In order to identify and characterize the underlying molecular mechanisms, we performed microRNA (miRNA) profiling of primary prostate cancers and non-cancer prostate tissue. Furthermore, using database analysis, we sought to identify regulatory targets of aberrantly expressed miRNAs. Material and Methods: We performed comparative miRNA expression profiling in ten prostate cancer specimens and ten non-cancer prostate tissue samples using deep sequencing of cDNA libraries. The deregulation of selected miRNAs was validated in established prostate cancer cell lines by Northern blotting as well as in 26 paired samples of prostate cancer tissue and adjacent normal tissue by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The regulative capabilities of selected miRNAs on potential target genes were analyzed in vitro using reporter gene constructs and transfection of miRNA expression vectors Results: Using deep sequencing we found that 16 miRNAs were up-regulated more than 1.5-fold and 17 miRNAs were down regulated more than 1.5-fold. The most pronounced deregulation was noted for miRNAs miR-375 and miR-200c which showed an up-regulation of more than 4.5-fold and miR-143 and miR-145 which were down regulated 4-fold each. The differential expression of these miRNAs could be confirmed by Northern blot analysis of established prostate cancer cell lines. By analyzing 26 primary prostate cancer samples by qRT-PCR we could detect a significant deregulation of the miRNAs miR-375, miR-143 and miR-145 (p Conclusion: Our findings indicate that specific miRNAs are reproducibly found to be deregulated in different cohorts of prostate cancer specimens using independent experimental methods. This is the first report to show that miRNAs miR-143 and miR-145 are capable of negatively regulating the protein expression of Myosin VI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4036.