| Bladder cancer is the most common malignant tumor in urogenital tract and the ninth common cancer in worldwide cancer incidence, with an estimated 386,300 new cases and 150,200 deaths in 2008. Although localized bladder cancers could be managed by surgical resection, the recurrence and progression rates are still high. In patients with advanced bladder cancer who receive radiotherapy or chemotherapy, the therapeutic outcomes remain unsatisfactory. The absence of more effective therapies for bladder cancer requires research into the underlying molecular mechanisms of bladder tumorigenesis and the development of new treatments aimed at specific molecular targets.MicroRNAs (miRNAs) are small (20-24nucleotides), endogenous RNAs that play important gene-regulatory roles by binding to the imperfectly complementary sequences at the 3’untranslated regions (3’-UTR) of mRNAs and directing their posttranscriptional repression. It is estimated that over 1000 miRNAs are encoded in the human genome, and they target approximately 60% of mammalian genes. The deregulation of miRNAs has been identified in various types of cancer including bladder cancer, which is involved in tumor cell proliferation, apoptosis, differentiation, angiogenesis, metastasis, and other functions. It is predictable that microRNA might be applied in the diagnosis and treatment of malignant tumors.Our research focused on the biological effect of miR-608 and its epigenetic regulation mechanism in bladder cancer. The main investigations and results are as follows:1) miR-608 was quantified in human bladder cancer tissue by qPCR. Out of 13 paired bladder cancer tissues and adjacent non-tumorous tissues, all cancer tissues presented with miR-608 in down-regulated pattern while comparing with normal adjacent tissue. The mean miR-608 expression level in cancer tissue was about 1/3 of that in normal adjacent tissue. However, no differences were detected when the bladder cancer cases were stratified by histological stage or grade.2) The results of BSP revealed that methylation levels were significantly higher in bladder cancer cell lines (T24 and UM-UC-3).3) To better characterize the role of miR-608 in bladder cancer, we conducted gain of function and loss of function analysis by transfecting bladder cancer cell lines (T24 and UM-UC-3) with chemically synthesized miR-608 mimic or inhibitor. We found that over-expression of miR-608 could induce G1-phase arrest in bladder cancer cells, and subsequently inhibit cells growth and colony formation. On the contrary, downregulation of miR-608 with specific inhibitor shows a totally reverse effect. However, no effect on cell apoptosis was detected and cell migration and invasion were undisturbed either.4) Subcutaneous UM-UC-3 xenograft was successfully establish in nude mice. Tumor growth was suppressed in miR-608 treated group. Down-regulation of Ki67 (proliferation related proteins) and FLOT1 were detected in miRNA treated group.5) We explored the possible targets of miR-608 by searching the TargetScan and miRanda online databases. We identified that FLOT1 could be novel direct target of miR-608. Further qPCR analysis and luciferase assay confirmed that miR-608 transcriptionally repressed FLOT1 by interacting with the essential binding sequence located in 3’-UTR.6) Further studies showed that miR-608 inhibited bladder cancer cells proliferation partly via AKT/FOXO3a pathway.In conclusion, we proved that miR-608 was able to suppress proliferation of bladder cancer cells by directly targeting FLOT1. |
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