Research On Regulatory Role Of MiR-218for Renal Cell Carcinoma By RAB6C

Background：Renal cell carcinoma(RCC) is a commom urologic tumor, and only afew cases have typical symptoms. There is no ideal markers for early diagnosis andprognosis of RCC after surgery in clinical practice. The effect of treatment is poor forrecurrence and advanced metastatic RCC. The efficacy of traditionl treatment such aschemotherapy and radiothrapy is not sensitive to RCC. Currently there is a need for newclinical biological markers for early diagnosis and prognosis of RCC, and as a new targetfor cancer gene therapy. Futhermore, it can help to improve the effect of treatment of RCC,especially for advanced RCC, and prolong survivalas a new target for cancer gene therapy,improving kidney cancer, especially in the treatment of advanced kidney cancer andprolong survival, and benefit for patients.Objective: In order to find new way to treat RCC, we need to understand themachanism of miRNA in RCC. To achieve this purpose, we need to detect the expressioncharacteristics of miR-218in RCC, and the regulatory role of RCC.Methods：1. To understand the expression of miR-218in RCC, we detect expression of miR-218in RCC and adjacent tissue using quantitative real-time PCR.2. Applicatioin of human Has-miR-218as a template to design primers, amplify thenucleotide sequence by PCR. MiR-218was cloned into the plasmid, and build a stableexpression plasmid of miR-218, and verify plasmid function in Rcc cell lines.3. Application of miR-218vector, transfect to RCC cell lines in vitro, and regulate theexpression level of miR-218to investigate biological effects of miR-218, such as changesin proliferation, invasion, apoptosis ability etc.4. Using bioinformatics, predict and analysis the target genes of miR-218. Usingdesined primer, clone target genes to fluorescein vector, and transfect to RCC cell lines,and detect the expression of fluorescent after cell disruption. 5. It investigate the regulatory roles of miR-218by RAB6C pathway in vivo, andshow inhibitory effect on RCC by up-regulation fo miR-218expression.Results：1. In this study, we tested the expression of miR-218of RCC and adjacent tissuesamples by using PCR method. We found that the expression levels of miR-218showedsignificantly lower expression, compared with normal tissues, suggesting that the miR-218may be a tumor suppressor gene in RCC.2. In this study, we constructed miR-218expression plasmid pcDNA3.1-miR-218successfully. This plasmid conform to requirement in design by detection and identification.This vector can be applied investigation of function and mechanism of miR-218.3. Application of miR-218expression vector, transfect to RCC cell lines, up-regulatethe expression of miR-218. Using standard test of tumor in vitro, found that it can inhibitRCC tumor cell growth, proliferation and invasion, and enhances cell apoptosis ability byup-regulated the expression level of miR-218.4. RAB6C is one of the target of miR-218by bioinfomatics prediction. It verified thatiRAB6C is the target of miR-218by fluorescein detection. The results of Western blottesting suggest that the expression of RAB6C is significantly decreased inmiR-218-transfected tumor cells. It shows that miR-218can inhibit proliferation andinvasion ability of tumor cells, and promote apoptosis of cells, by regulated expression ofRAB6C.5. In vivo, incresed the expression of miR-218, the expression of RAB6C isdown-regulation, and it can inhibit the growth of RCC.Conclusions：In these experiment, we know that miR-218has a regulatory role in cellgrowth, proliferation, invasion and apoptosis to RCC cells. Its regulatory role is achievedby regulating RAB6C. Increased the expression of miR-218, can inhibit the growth ofRCC tumor in vivo and vitro. It can provides a new way for tumor biotherapy of RCC.