Research Of Survivin Functions In Transitional Cell Carcinoma Of Bladder By RNA Interference

Objective To identified the plasmid containing short hairpin RNA(shRNA)of survivin .Methods PshRNA-survivin expression plasmid get from Dr Huang Ai-long;pshRNA-survivin was emplified in system and identitified by enzyme digestion and sequencing method.Results The recombinant plasmid pshRNA-survivin was identified successfully by enzyme digestion and sequencing.Conclusion The results show that the short hairpin RNA of survivin can be efficiently identified. Objective To observe the plasmid containing short hairpin RNA(shRNA)of survivin suppressed the expression of exogenous survivin gene in T24 cells.Methods The plasmid pshRNA-survivin was stably transfected into TCCB cells T24 to detect effect of survivin expression and analyze the inhibition of survivin gene with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot. Results The expression of surviving in transfected T24 cells was markedly depressed at both mRNA and protein levels (61.73%, 53.37%, respectively) as compared with control.Conclusion The results showed that down-regulate the expression of survivin with RNA interference technology can significantly suppress expression of survivin to a certain degree. Objective To assess the effects of pshRNA-survivin on the biological behaviors of T24 cells by gene siliencing.Methods The biological effects were observed, including anchorage-independent growth by MTT method, and the effects of apoptosis inducing on T24 cells were detected by flow cytometry assay and comfired by electron microscope, the ability of mobility and invasion in vitro by cell migration assay and tranwell chamber assay.Results The growth of tumor cells was retared by anchorage-independent growth assay. The inhibitory percentages of T24 cells of the experimental groups correlated with the negative control groups were 15.69%,27.64%,59.13% in 24hour,48hour and 72hour, respectively. Meanwhile, 24h after transfection, it was observed that silencing survivin by RNAi could significantly induce the spontaneous apoptosis of T24 cells at the rate 16.76±1.31%, detected by flow cytometry assay and comfirmed by electron microscope. In addition, fewer penetrating cells of pshRNA-survivin group were observed along with a marked inhibition of invasion by tranwell chamber assay (27.62±2.06,26.07±1.73,10.34±1.85*, respectively).Conclusion The results showed that blocking the expression of survivin with RNA interference technology can significantly suppress expression of proliferation,mobility and invasion of T24 cells, and induce apoptosis to a certain degree. RNAi targeted to survivin has a potential value in gene therapy of bladder cancer. Objective To observe the effects of pshRNA-Survivin on human bladder cancer T24 cell line and to suppress the expression of survivin gene in the animal model of BALB/c xenograft tumor.Methods Establishment of TCCB T24 lines with expressing siRNA-survivin stably. Then T24 cells were inoculated into the right-back legs of BALB/c nude mice to establish bladder cancer model. The inhibitory effects were observed on the growth of tumor and analyzed the inhibition of survivin gene with SABC of immunohistochemistry; Microscope were used to observe the morphological changes.Results Nude mice were injected subcutaneously with siRNA-survivin cells and xenograft tumor formed 17.0 days later,no subcutaneous metastasis.While the other two groups formed tumor at 12.4days,11.8days,respectively,six/all with subcutaneous metastasis.Tumor size were measured with caliper every seven days.21days after formed xenograft tumor,mice were sacrificed and their tumors were weighted for evaluative antitumor ratio.Result showed that the volume of T24/ siRNA-survivin xenografts in mice was effectively inhibited by pshRNA-survivin.The Ratio of antitumor was significantly higher in pshRNA-survivin group than those in control groups. The expression of survivin in pshRNA-survivin group in vivo was markedly depressed as compared.Conclusion pshRNA-survivin exerts its antitumor effect on human bladder cancer T24 cell lines in vivo. The mechanisms may be changing tumor cell cycle and inducing tumor cell apoptosis by down-regulating the expression of survivin.