| Background: Urinary bladder cancer is among the most common tumors in urinary tract. According to the statistics of National Cancer Institute, 68810 new cases were diagnosed and 14100 patients died in 2008, costing $2.9 billion annually. Unfortunately, patients with advanced disease face a 5 year survival rate of approximately 20–40% despite wide range of treatment modalities including surgery, chemotherapy and radiotherapy. Recent advances in molecular pathology have revealed that bladder cancer is a disease of multiple gene aberrations, including amplification and overexpression of oncogenes and mutation or deletion of tumor suppressor genes, which result in the deregulation of signaling transduction pathways, leading the tumor cells to escape from the normal growth control mechanism and present the malignant phenotype of invasiveness and increased angiogenesis. microRNAs (miRNAs or miRs), 21-25 nucleotides in length, are a recently discovered class of non-coding RNAs. Through the specific binding to the 3'UTRs of mRNAs, miRNAs down-regulate gene expression by either inducing degradation of target mRNAs or impairing their translation. Some studies demonstrated that more than 50% of miRNA genes are located in cancer associated genomic regions or in fragile sites of chromosomal regions. Numerous evidences point to a role for miRNAs in the etiology and pathogenesis of cancer by targeting oncogenes or tumor suppressors. Some microRNAs were demonstrated to be dysregulated in diverse cancer subtypes including hepatocellular carcinoma, lung cancer, breast cancer, colorectal cancer and other cancers. However, the expression and function of microRNAs in bladder cancer are still scarce.Aim of this study: To find the dysregulated microRNAs in bladder cancer by microRNA array and verify some of these differentially expressed microRNAs in a enlarged sample population by realtime RT-PCR. Furthermore, to explore the functional aspects of some microRNAs in bladder cancer cell lines in vitro.Methods:1. microRNA array screening and verifying. We collected 3 pairs of bladder cancer tissues and its para-tumor control. small RNA was extracted using mirVANA microRNA extraction kit. RNA was labeled by Cy3 and hybridized with Agilent microRNA array. After incubation and washing, the array was scanned by Agilent scanner, raw data was obtained using Feature Extraction software and further analyzed. The results were displayed as the ratio of the microRNA signal in tumor to that in para-tumor tissues. Then cluster analysis was done by cluster 3.0 software using selected data. We verified part of the dysregulated microRNAs in a total 26 samples by realtime RT-PCR, which included 20 tumor tissues and 6 normal bladder mucosae. Total RNA was extracted using TRIzol according to protocol, then reversely transcribed using gene specific stem-loop RT primer. The cDNA was then quantified by a taqman probe based relative quantitative study using RNU6B as the internal control. The relative gene expression was calculated and compaired using student t test. 2. Functional study of microRNA 125b?Relative gene expression was examined in bladder cancer cell lines J82, EJ and T24 by realtime RT-PCR method as previous. Transient transfection with miR-125b mimics or its antisense inhibitor was performed in T24 cells using liposome based transfection reagent. Cell proliferation was evaluated using MTT assay. And in vitro clone-formation assay was performed. ERBB2, one of the targets of miR-125b was assessed at mRNA and protein level using semi-quantitative RT-PCR and western blot respectively. 3. Functional study of microRNA 21. Transient transfection with miR-21 mimics or its antisense inhibitor was performed to assess its influence to cell proliferation and clone formation in T24 cells. IC50 of T24 cells to a cytotoxic agent doxorubicin was calculated using a MTT based method. Doxorubicin induced cell apoptosis was evaluated by Hoechst 33324 dyeing. BCL2, a major anti-apoptosis protein in cells was assayed by western blot. 4. Functional study of microRNA 221. Northern blot analysis was used to determine miRNA-221 expression levels in bladder cancer T24 cells, RT4 cells and human normal urothelial cells. miR-221 was silenced with antisense oligonucleotides in T24 cells and pro-apoptotic effect of necrosis factor related apoptosis-inducing ligand (TRAIL) on miRNA-221-silenced cells was assessed with flow cytometry. The p27kip1 protein expression in miRNA-221-silenced cells exposed to TRAIL was detected by Western blotting. The role of miR-221 silencing on T24 cell cycle phase distribution was investigated through flow cytometric analysis.Results: 1. Multiple microRNAs were found by microRNA array screening, in which miR-141, miR-200c, miR-96, miR-182, miR-183 and so on were up-regulated over 3 folds while miR-125b, miR-143, miR-145, miR-99a, miR-100, et al were down-regulated over 3 folds. miR-125b, miR-143, miR-145 were significantly down regulated when verifying in larger samples using realtime RT-PCR. Furthermore, the expression of miR-143 and miR-145 were highly related. 2. The expression of miR-125b was low in bladder cancer cell lines, especially in T24 and EJ cells. Transfection with synthetic miR-125b mimics could significantly increase the miR-125b level in T24 cells, while antisense oligonucleatides inhibitor of miR-125b could lower the level of miR-125b. when treated with miR-125b mimics, T24 cells showed a 15.9%, 25.0%, 18.8% proliferation sirpression rate in 48, 72, and 96 hours. Clonal formation was also repressed after miR-125b mimics transfection. Furthermore, miR-125b mimics could down-regulate ERBB2 in mRNA and protein level while anti-miR-125b could up-regulate it. 3. miR-21 transfection induced little change in cell proliferation in T24 cell lines. However, inhibition miR-21 by antisense inhibitor surpressed cell proliferation. miR-21 mimics could also induce T24 cell resistance to doxorubicin, while anti-miR-21 could sensitizing cells to this cytotoxic drug. Doxorubicin induced cell apoptosis was significantly surpressed when treated with miR-21 mimics, but enhanced by anti-miR-21 treatment. BCL2 protein was up-regulated with miR-21 mimics transfection and vise versa. 4. Human miR-221 was significantly up-regulated in bladder cancer T24 cells and RT4 cells compared to human normal urothelial cells. T24 cell was TRAIL-resistant cell line. miR-221 silencing predisposed T24 cells to undergo apoptosis induced by TRAIL and resulted in an up-modulation of cyclin-dependent kinase inhibitor p27Kip1. miR-221 suppression promoted the activation of caspase 3 induced by TRAIL in T24 cells.Conclusions:1. Multiple microRNAs are dysregulated in urinary bladder cancer, including miR-143, miR-145, miR-125b, et al. 2. miR-125b surpress proliferation and clonal formation of bladder cancer cell line T24 in vitro and down-regulate expression of ERBB2 in both mRNA and protein level. 3. Down-regulation of miR-21 by antisense oligonucleotides renders proliferation inhibition in T24 cells and sensitizes T24 cells to doxorubicin through a BCL2 related pathway, enhancing doxorubicin induced cell apoptosis. 4. Down-regulation of miR-221 by antisense oligonucleotides sensitizes T24 cells to TRAIL induced cell apoptosis and increases expression of p27kip1, which surpresses cell cycle progression.These results demonstrate that microRNAs may participate in bladder cancers and may be a therapeutic target in the future. |
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