Effect Of MicroRNA-494Over-expression And RNA Interference-mediated Survivin Gene Inhibition On Xenograft Prostate Cancer Growth In Nude Mice

Objective:To study the effect of microRNA-494over-expression and RNA interference-mediated survivin gene inhibition on xenograft prostate cancer growth in nude mice.Methods:Ad-pre-microRNA-494(Ad-494) which can overexpressed microRNA-494and Ad-survivin shRNA (Ad-sur) which is loaded with survivin short hairpin RNA(shRNA) has has been previously constructed and proved to be effective by our departments. These two adenovirus were transfected HEK-293T cells respectively to produce target virus. After the target virus purified and determinated, PC-3cells were transfected according to the following group:PBS group(blank control),Ad-GFP group(negative control),Ad-sur group,Ad-494group,Ad-sur+Ad-494group (each group contain five nude mice). And a total of1×107cells were injected to the right armpit of the nude mice to establish xenograft prostate cancer model in nude mice.The gross tumor volumes were measured dynamically The mice were sacrificed and tumors were obtained55days after the injection. Tumor volume was calculated, Morphology of the tumor were determined by HE staining. The expression status of survivin gene in tumor sample were detected by western blot. Meanwhile, the expression of survivin,Bax,Bcl-2,p53,Caspase-3in tumor sample were detected by immunohistochemistry.Results:The titer of the adenovirus can be up to109pfu/mL. The best transfected dose is50MOI. The results from nude mice xenograft model showed that the tumor growth of in Ad-sur group、Ad-494group and Ad-sur+Ad-494group were retarded significantly. The tumor volume in the Ad-sur+Ad-494group were the smallest as compared to that in either Ad-sur group or Ad-494group (P<0.05), This mean it have synergetic anti-tumor effect. Immunohistochemistry and western blot showed that the expression status of survivin gene in Ad-sur group, Ad-494group and Ad-sur+Ad-494group were down-regulated apparently, which is more obviously in Ad-sur+Ad-494group. Ad-sur, Ad-494and Ad-sur+Ad-494can up-regulate Bax、p53、Caspase-3expression and down-regulate Bcl-2expression This is more apparent in the Ad-sur+Ad-494groupConclusion:Both Ad-pre-microRNAs-494and Ad-survivin shRNA can significantly inhibit the expression of surviving gene,and can retard the growth of xenograft prostate cancer tumor. The effect of Ad-sur+Ad-494were more obviously as compared with other groups. This indicated that target multiple site of a single gene is more effective. It may become a new gene therapy strategy for advanced prostate cancer. MicroRNA-494down-regulates survivin gene is associated with up-regulation of, Bax,p53,Caspase-3and down-regulation of Bcl-2.