miR-193b is an epigenetically regulated putative tumor suppressor in prostate cancer Hanna E. Rauhala 1 , Sanni E. Jalava 1 , Jarkko Isotalo 1 , Hazel Bracken 1 , Saara Lehmusvaara 1 , Teuvo L.J. Tammela 2 , Hannu Oja 3 and Tapio Visakorpi 1 1 Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland 2 Department of Urology, University of Tampere and Tampere University Hospital, Tampere, Finland 3 Tampere School of Public Health, University of Tampere, Tampere, Finland miRNAs have proven to be key regulators of gene expression and are differentially expressed in various diseases, including cancer. Our aim was to identify epigenetically dysregulated genes in prostate cancer. We performed miRNA expression profiling after relieving epigenetic modifications in 6 prostate cancer cell lines and nonmalignant prostate epithelial cells. Thirty-eight miRNAs showed increased expression in any prostate cancer cell line after 5-aza-2 0 -deoxycytidine (5azadC) and trichostatin A (TSA) treatments. Six of these also had decreased expression in clinical prostate cancer samples compared to benign prostatic hyperplasia. Among these, miR-193b was methylated in 22Rv1 cell line at a CpG island ~1 kb upstream of the miRNA locus. Expressing miR-193b in 22Rv1 cells using pre-miR-193b oligonucleotides caused a significant growth reduction (p < 0.001) resulting from a decrease of cells in S-phase of the cell cycle (p < 0.01). In addition, the anchorage independent growth was partially inhibited in transiently miR-193b-expressing 22Rv1 cells (p < 0.01). Altogether, our data suggest that miR-193b is an epigenetically silenced putative tumor suppressor in prostate cancer. Micro-RNAs (miRNAs) are a group of small, about 22 nucle- otide long nonprotein coding RNAs that were first identified in C. elegans, and since have been found to be key regulators of gene expression in other organisms as well. 1,2 miRNAs are first synthesized as long precursors called pri-miRNAs. These multi-hairpin structures are processed by a nuclear enzyme called Drosha, producing single hairpin molecules, pre-miR- NAs. Pre-miRNAs are exported from the nucleus into cyto- plasm where they are further cleaved into their mature dou- ble stranded RNA (dsRNA) form by Dicer and loaded into RNA-induced silencing complexes (RISC). One strand of this dsRNA becomes degraded leaving the active miRNA ready to bind to its target sequence in mRNAs. Depending on the level of complementarity of the miRNA to its target mRNA 3 0 -end untranslated region (3 0 -UTR) sequence, miRNA bind- ing can result in mRNA degradation or translational repres- sion (reviewed in Ref. 3). Since 1 miRNA can target various mRNAs and 1 mRNA can be targeted by several miRNAs, these small regulators add a new complex layer on gene expression control. Since the discovery of miRNAs, there have been numerous reports indicating altered miRNA expression in various dis- ease states, such as cancer, cardiovascular disease and diabe- tes. In fact, miRNA expression profiling has proven to be more accurate in identifying tumor subcategories and pre- dicting clinical outcome than classical mRNA profiling. 4 Many miRNAs are located in chromosomally unstable regions making them prone to aberrations. 5 Reports have indeed shown copy number alterations at miRNA loci lead- ing to increased or decreased miRNA copy number. 6–8 miRNA genes have also been found to harbor mutations. 6 All in all, miRNAs undergo the same spectrum of genomic aberrations in cancer as do protein-coding genes. Similarly to protein-coding genes, miRNAs are transcribed by RNA polymerase II, and the same intrinsic regulatory sys- tems are involved in their initial expression as in the expres- sion of mRNAs. Also, the expression of miRNAs can be regulated through epigenetic mechanisms, i.e., DNA methyla- tion and histone methylation and acetylation. Several tumor suppressive property possessing miRNAs, such as miR-34a targeting E2F3 and BCL-2, miR-124a targeting CDK6, and miR-127 targeting BCL-6, have been shown to be epigeneti- cally silenced through DNA methylation in various cancer types, both in cancer cell lines and in clinical tumors. 9–11 In addition to miRNAs being targeted by epigenetic Key words: prostatic carcinoma, neoplasia, CpG, methylation, miRNA Additional Supporting Information may be found in the online version of this article. Grant sponsor: European Community’s Seventh Framework Programme; Grant numbers: FP7/2007-2013, HEALTH-F2-2007- 201438; Grant sponsors: Academy of Finland, Cancer Society of Finland, Reino Lahtikari Foundation, Sigrid Juselius Foundation, Competitive Research Funding of the Pirkanmaa Hospital District, Finnish Cultural Foundation DOI: 10.1002/ijc.25162 History: Received 19 Aug 2009; Accepted 17 Dec 2009; Online 13 Jan 2010 Correspondence to: Tapio Visakorpi, Institute of Medical Technology, FIN-33014 University of Tampere, Tampere, Finland, Tel: þ358-3-3551-7725, Fax: þ358-3-3551-8597, E-mail: tapio. visakorpi@uta.fi Cancer Genetics Int. J. Cancer: 127, 1363–1372 (2010) V C 2010 UICC International Journal of Cancer IJC