PDF file - 325K, Supplemental Figure 1: Downregulation of miR-34b expression, its potential diagnostic and prognostic significance and methylation status in PCa. A) Box plot representation of mir-34b expression in a cohort of unmatched tissue samples. B) Box plot representation of ISH results of tissue samples. (Ave- Average; T/N- Tumor/Normal). C) Chi-square test showing correlation of clinicopathological characteristics with miR-34b expression. D) CpG islands within the 1.0 kb region upstream of the miR-34b gene. Arrow indicates the start of the miR-34b gene. Locations for primers and probes are also shown on the miR-34b gene. F- Forward, R-Reverse, M/U Methylation and Unmethylation probes, CF1, CF2, CR1, CR2- ChiP Forward1-2, ChIP Reverse 1-2. E) Correlation of miR-34b expression with average percent methylation indicating cases with low miR-34b expression have higher methylation and vice versa.
ARTICLE ABSTRACT
Purpose: miRNAs can act as oncomirs or tumor-suppressor miRs in cancer. This study was undertaken to investigate the status and role of miR-34b in prostate cancer.Experimental Design: Profiling of miR-34b was carried out in human prostate cancer cell lines and clinical samples by quantitative real-time PCR and in situ hybridization. Statistical analyses were done to assess diagnostic/prognostic potential. Biological significance was elucidated by carrying out a series of experiments in vitro and in vivo.Results: We report that miR-34b is silenced in human prostate cancer and the mechanism is through CpG hypermethylation. miR-34b directly targeted methyltransferases and deacetylases resulting in a positive feedback loop inducing partial demethylation and active chromatin modifications. miR-34b expression could predict overall and recurrence-free survival such that patients with high miR-34b levels had longer survival. Functionally, miR-34b inhibited cell proliferation, colony formation, migration/invasion, and triggered G0/G1 cell-cycle arrest and apoptosis by directly targeting the Akt and its downstream proliferative genes. miR-34b caused a decline in the mesenchymal markers vimentin, ZO1, N-cadherin, and Snail with an increase in E-cadherin expression, thus inhibiting epithelial-to-mesenchymal transition. Finally we showed the antitumor effect of miR-34b in vivo. MiR-34b caused a dramatic decrease in tumor growth in nude mice compared with cont-miR.Conclusion: These findings offer new insight into the role of miR-34b in the inhibition of prostate cancer through demethylation, active chromatin modification, and Akt pathways and may provide a rationale for the development of new strategies targeting epigenetic regulation of miRNAs for the treatment of prostate cancer. Clin Cancer Res; 19(1); 73–84. ©2012 AACR.
