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Martin Hart,
Sven Wach,
Elke Nolte, Jaroslaw Szczyrba,
Roopika Menon,
Helge Taubert,
Arndt Hartmann,
Robert Stoehr,
Wolf Wieland,
Friedrich A Grässer,
Bernd Wullich
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ABSTRACT: Prostate cancer (PCa) is a leading cause of cancer mortality in men. One of the distinct characteristics of PCa is the over-expression of the ERG proto-oncogene. The TMPRSS2-ERG gene fusion, the most common gene fusion, is found in approximately 50% of PCa cases. We show that certain microRNAs (miRNAs) are extensively deregulated in PCa cell lines and primary clinical cancer samples. MiRNAs are capable of modulating post-transcriptional gene expression via inhibition of protein synthesis. Independent target prediction methods have indicated that the 3' untranslated region (3'UTR) of the ERG mRNA is a potential target of miR-145. MiR-145 is consistently downregulated in PCa. Here we show that the ERG 3'UTR is a regulative target of miR-145 in vitro. Ectopic expression of miR-145 led to a reduction in the expression of the ERG protein. We analyzed 26 prostate cancer samples with their corresponding normal tissue. ERG protein expression was found to be elevated in the tumor samples, along 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 downregulation of miR-145 might contribute to the increased expression of most ERG splice variants sharing the miR-145 target sequence in their 3'UTR. © 2013 The Authors Journal compilation © 2013 FEBS.
FEBS Journal 03/2013; · 3.79 Impact Factor
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Jaroslaw Szczyrba,
Elke Nolte,
Martin Hart,
Celina Döll,
Sven Wach,
Helge Taubert,
Bastian Keck,
Elisabeth Kremmer,
Robert Stöhr,
Arndt Hartmann,
Wolf Wieland,
Bernd Wullich,
Friedrich A Grässer
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ABSTRACT: 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.
International Journal of Cancer 07/2012; · 5.44 Impact Factor
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ABSTRACT: 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.
Molecular Cancer Research 06/2011; 9(6):791-800. · 4.29 Impact Factor
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Sven Wach,
Elke Nolte, Jaroslaw Szczyrba,
Robert Stöhr,
Arndt Hartmann,
Torben Ørntoft,
Lars Dyrskjøt,
Elke Eltze,
Wolf Wieland,
Bastian Keck,
Arif B Ekici,
Friedrich Grässer,
Bernd Wullich
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ABSTRACT: 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.
International Journal of Cancer 03/2011; 130(3):611-21. · 5.44 Impact Factor
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Jaroslaw Szczyrba,
Elke Löprich,
Sven Wach,
Volker Jung,
Gerhard Unteregger,
Stephanie Barth,
Rainer Grobholz,
Wolf Wieland,
Robert Stöhr,
Arndt Hartmann,
Bernd Wullich,
Friedrich Grässer
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ABSTRACT: 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.
Molecular Cancer Research 03/2010; 8(4):529-38. · 4.29 Impact Factor
