A Study Of The MiR-137 Expression And Its Impact On The Proliferation And Invasion Of Bladder Cancer

Bladder cancer is the fifth most common cancer in the general population and the second most common cause of death in patients with genitourinary tract malignancies. More than 90% of urinary bladder tumors are comprised of transitional cell carcinoma(TCC) that arises from transitional epithelium. Bladder tumors can be classified into two distinct categories: non-muscle and muscle invasive bladder cancer. About 80% patients with bladder cancer were early diagnosed with non-muscle invasive bladder cancer,while in the later they got the positive bladder endoscopic surgery and bladder perfusion therapy, but the proportion of patients with recurrence in 5 years still account for 50%-70%; And about 10%- 30% patients were further developed into muscular invasion urothelial carcinoma. Invasive urothelial carcinoma has strong ability of attack, the patient’s prognosis is poore rand the 5-year survival rate is low. Therefore, exploring the molecular mechanism of bladder cancer development, and looking for a reliable biomarker and effective therapeutic targets to improve the survival rates of patients with bladder cancer is particularly important.Recently, increasing evidences suggest that a new class of RNAs, known as micro RNAs(mi RNAs), could regulate various target genes, including oncogenes and tumor suppressor. mi RNAs are endogenous 18~24 nt non-coding RNAs, which can negatively regulate the protein expression by specifically binding to the 3’untranslated regions(3’UTR) of target m RNAs. A lot of research studies have demonstrated that mi RNAs contribute to most basic biological processes, such as development, differentiation, apoptosis and cell proliferation. mi RNAs have been reported to relate to the progression of a variety of tumor, such as stomach cancer, lymphoma, liver cancer, lung cancer, endometrial cancer. mi R-137 has attracted much attention because it is frequently down-regulated and functions as a tumor suppressor in many cancers such as ovarian cancer, gastric cancer, glioblastoma, lung cancer, colorectal cancer and neuroblastoma. Previous study has showed that mi R-137 was frequently methylated in primary tumors and ectopic expression of mi R-137 suppressed bladder cancer cell proliferation. However, the expression and function of mi R-137 in the bladder cancer remains poorly understood previously. In this study, we mainly explore the role of mi R-137 in bladder cancer.In this study, we used the mi RNA chip of Agilent company to analyse the the mi RNA expression between the bladder cancer tissue and normal bladder tissues adjacent to carcinoma, which has a high throughput, high sensitivity and high specificity. The data obtained was analyzed by Agilent Feature Extraction and 10.5 genespring. The mi R-137 highly expressed in in bladder cancer was choosen as the research object. At the same time, the structure and expressing characteristics of mi R-137 are analyzed by using biological information. The results showed that the expression mi R-137 may be regulated by the epigenetic modifications DNA methylation. The expression of mi R-137 was first evaluated by quantitative real-time-PCR(q RT-PCR) in bladder cancer cell lines and one normal bladder cell line SV-HUC-1. mi R-137 was significantly up-regulated in all the bladder cancer cell lines compared with SVHUC-1. We further quantified the expression level of mi R-137 in 6 pairs of human bladder cancer tissues and adjacent normal mucosal tissues by q RT-PCR. The results showed that the expression level of mi R-137 was generally higher in tumor tissues compared to matched non-tumor tissues. To study the relationship of mi R-137 with bladder cancer occurrence, the expression of mi R-137 was detected in 50 clinical patients using q RT-PCR. Out of 50 bladder cancer samples, mi R-137 was up-regulated in 45 cases(45/50, 90%) compared with adjacent tissues. Meanwhile, mi R-137 was down-regulated in 5 cases(5/50, 10%). In general, the expression of mi R-137 in bladder cancer tissues was significant higher than in adjacent tissues. The higher level expression of mi R-137 was was associated with p TNM stage, and higher level of mi R-137 was associated with p M stage(p≤0.05, metastasis vs. no metastasis) in bladder cancer patients. These data suggested that alterations of mi R-137 could be involved in bladder cancer progression. The potential impact of mi R-137 in bladder cancer cell prolifetation, migration and invasion were detected. Cells were transfected with scrambled control oligo or mi R-137 mimics and inhibitor, which showed high transfection efficiency. CCK-8 proliferation assay showed that cell proliferation was promoted in mi R-137 mimics-transfected bladder cancer cells compared with scrambled oligo-transfected cells or untreated cells. Conversely, mi R-137 inhibitor significantly inhibited the cell proliferation of the T24 cells. Intriguingly, migration and invasion assay showed that overexpression of mi R-137 significantly promoted the migration and invasion of T24 cells compared with the control, whereas adding mi R-137 inhibitor inhibited both cell migration and invasion.As predicted by Pictar, there was complementarity between has-mi R-137 and the PAQR3 3’UTR. Overexpression of mi R-137 reduced the protein and m RNA levels of PAQR3 in bladder cancer cells. The effect of mi R-137 on PAQR3 protein was then assessed by using a luciferase reporter assay. The results indicates that mi R-137 directly targeted the PAQR3 3’UTR. We first analyzed the function of PAQR3 in bladder cancer cells. Transfection of small interfering RNA against PAQR3 into T24 cells led to dramatically decreased PAQR3 protein expression. Silencing of PAQR3 significantly enhanced the proliferation of bladder cancer cells, which phenocopied the effects of mi R-137 on proliferation of bladder cancer cells. Cotransfection of a construct expressing PAQR3 and mi R-137 in T24 cells result in the restoration of PAQR3 expression, as confirmed by Western blot. This expression of PAQR3 significantly inhibited the proliferation and invasion of bladder cancer cells. More importantly, we found that expression of PAQR3 could significantly reverse the proliferation and invasion promotion imposed by mi R-137. In summary, these data indicate that inhibition of PAQR3 expression by mi R-137 is responsible, at least in part, for the mi R-137 promotion of cell proliferation and invasion in human bladder cancer.In conclusion, our results have showed that mi R-137 was significantly upregulated in bladder cancer cells and tissues. Overexpression of mi R-137 promoted bladder cancer cell proliferation and invasion through directly targeting PAQR3. These findings suggest that this novel mi R-137/PAQR3 axis provides new insight into the mechanisms underlying tumor metastasis.