The Experimental Study On The RNA Interference Targeting Survivin Gene Suppresses Bladder Cancer Growth

【Objective】Bladder cancer is the most common malignancy of the genitourinary tract in China, and the majority of tumors are superficial at the time of diagnosis. Intravesical chemotherapy and immunotherapy is the standard approach for patients after transurethral resection of bladder tumor (TURBT) in order to prevent tumor recurrence. As the high odds of recurrence, we need new methods for intravesical treatment. Survivin gene is a member of inhibitor of apoptosis proteins(IAPs), and has become an important target for gene therapy. RNA interference is a high effective method for gene silence. To continue former research, we studied RNA interference targeting Survivin gene suppress bladder cancer cell T24 growth in vivo and therapeutic effect in orthotopic bladder cancer model.【Methods】(1) Survivin-targeting shRNA eukaryotic expression vector pRNAT-survivin was constructed and identified. Human bladder cancer cell T24 was transfected with pRNAT-survivin and then stable monoclonal cells were selected. Survivin mRNA expression of transfected was detected by real time PCR. Then stably transfected and untransfected cells were inoculated respectively into subcutaneous tissue of nude mice, and tumor growth status were recorded. Survivin protein expression of tumors were investigated by immunohistochemistry. (2) The mucosa was mechanically damaged transurethrally under euthyphoria, and human bladder cancer cell T24 was instilled into bladders of 25 BALB/c nude mice, to establish orthotopic bladder cancer model. We utilized MRI to monitor tumor progression, and Gd-DTPA was used as contrast agent .The bladders of nude mice were stained with HE for pathologic examination. (3) We established orthotopic bladder cancer model with the same method. Mice were randomly divided into 4 groups, six mice each group, group A: untreated; group B: intravesical treatment with mitomycin c at the concentration 0.5 mg/ml, 0.06ml once, twice a week; group C: intravesical treatment with plasmid RNAi-survivin at the concentration 150μg/ml, 0.06ml once twice a week; group D: intravesical treatment with plasmid and MMC in turn, twice a week. The bladders were removed and weighed after treatment for 6 times. The expression of Survivin and caspase-3 was examined by immunohistochemistry.【Results】(1)The shRNA eukaryotic expression vector was successfully constructed. The expression efficiencies of transfected cells was 94.7% after selection and choose monoclonal cells. Survivin mRNA expression of the stably tansfected cells downregulated 92.86% compared with untransfected cells. The tumor volum of the untransfected group exceeded 2000 mm3 between 20 and 29 days after inoculation, but the transfected group had only three tumors no more than 62.5 mm3 after additional 2 weeks. Survivin protein expression of the tumors in transfected group was weaker than that in untransfected group. (2) All the 25 animals established bladder cancer after inoculation. Seven days after inoculation pathologic changes but not MRI abnormalities were found in bladders of nude mice.14,21 and 28 days after inoculation MRI could detect filling defect in all the animals and correlated well with actual tumor size. Pathologic examination showed: tumor grew in the mucosa(surface) of bladder 7 days after inoculation;limited in muscle between 14 and 28 days; invaded serosa after 35 days. (3) The tumor weight of group A,B,C,D were 62.48±5.14mg,41.58±7.90mg,38.60±4.44mg,26.97±6.19mg respectively. RNAi plasmid targeting Survivin gene at the concentration of 150μg/ml got the similar anti-tumor effect with MMC at the concentration of 0.5mg/ml after intravesical treatment. While combining the two for instillation in turn were more effectively. Immunohistochemistry demonstrated that the whole tumor of group A was Survivin strong positive expression and caspase-3 protein little expression; Survivin protein expression of superficial layer tumor cells in group C and D become weaker; caspase-3 protein was expressed in superficial layer tumor cells in group B,C and D, and rarely in deep layer. 【Conclusion】(1) The application of siRNA-survivin could markedly inhibit ectopic tumor growth. The shRNA targeting Survivin lastingly inhibited bladder cancer cell T24 growth in vivo. Survivin may act as a valuable target for gene therapy. (2) We successfully established an orthotopic bladder cancer model, which simulated progression of human bladder cancer approximately. MRI was an reliable way for dynamic detection of murine orthotopic bladder tumor with optimized Gd-DTPA concentration. (3) RNA interference expression plasmid targeting Survivin has similar effect compared with MMC, and showed synergistic effect when combined with MMC. The mechanisms may be that suppressing Survivin expression of bladder tumor provoke cancer cells to apoptosis, and then effect tumor growth; the synergistic effect may be explained by their effect in different cell circle, and increase chemosensitivity of bladder cancer to MMC. In summary: In the research we noticed the therapeutic effect of RNA interference targeting Survivin gene in vivo. Combining this with conclusions in former research, we consider Survivin gene maybe a potential target for gene therapy. But the mechanism of action and the approach to apply needs more study.