Inhibition Of Renal Cancer Growth In Nude Mice By SiRNA Targeting On Survivin

Renal cancer wich is an abbreviation for renal cell carcinoma (RCC) and renal adenocarcinoma, is a malignant tumor which derives from renal tubule urinary system. Rcc is one of the malignant tumors which is secondary to bladder cancer in the urinary carcinoma, and renal cell carcinomas(RCCs) of the clear-cell type is the majority tissular type of RCC.RCC has more malignant and insensitivity to radiotherapy.There are some specific medicines which maybe cure RCC, but some problems also exist. This urge us to seek new methods for treatment.Today the gene therapy to RCC has been the hotspot in tumor therapy.Survivin gene is a new member of inhibitors of apoptosis protein(IAP) family.It is observed express mainly in the common human malignant tumor tissues,but not in the adult differentiated tissues. Survivin gene's selective expression in tissues suggests that it is likely to be identified as a special index in diagnosis and a potential therapeutic target in RCC.We have transfected shRNA-survivin with a vector of pGenesil-1 into human renal cancer cell line 786-0. ShRNA-survivin effectively suppressed the expression of survivin in mRNA and protein level and the activity of 786-0.All of this give us basic data of RNAi targeting survivin in therapy of human renal cancer in vitro.In this study,on the basis of survivin's high expression in 786-0. We injected shRNA-survivin with a vector of pGenesil1.1into the xenograft tumor in nude mice inoculated by 786-0.Tumor volumes in each group were calculated every day. After all the mice were sacrificed and the tumors were taken out to undergo RT-PCR and Western blot to detect survivin expression. Tumor cell apoptosis was detected by TUNEL. All of this give us basic data in gene therapy of RCC. PART ONEEXPRESSION AND SIGNIFICANCE OF SURVIVIN GENE IN HUMAN RENAL CARCINOMA 786-0 CELLSObjective:The study was designed to examine the expression of survivin gene in human renal carcinoma 786-0 cells,and then discussed its significance.Methods: We cultured human renal carcinoma 786-0 cells. RT-PCR and Western blot were used to detect survivin expression in the cell line.Results: RT-PCR and western blot showed that survivin gene was expressed in human renal carcinoma 786-0 cells.Conclusions:Survivin gene was expressed in human renal carcinoma 786-0 cells,which suggested that survivin gene may play an important role in the development of RCC. This also gives us basic data in gene therapy of RCC. PART TWO THE CONSTRUCTION AND AMPLIFICATION OF SURVIVIN-SPECIFIC SIRNAObjective:According to survivin gene sequences in Genebank, two groups of survivin-specific siRNA were constructed. The optimal conditions for the expressions were identified.Methods: According to survivin gene sequences in Genebank,two groups of survivin- specific siRNA constructed.We designed two masculine siRNA and a negative one.Plasmid pGenesil1.1 was connected with them,and they were transformed in DH5α.The coenobium of anti-kanamycin was chosen and cultured.And then,we assayed these siRNA.Results: We got two masculine siRNA(survivin1 5′-GGACCACCGCATCTCTACA-3′166; survivin2 5′-GGCTGGCTTCATCCACTGC-3′241) and one negative siRNA(HK 5′-GACTTCATAAGGCGCATGC-3′).Conclusions: The survivin-specific siRNA which was designed in vitro offered a valuable arm for us to restrain expression of survivin in tumor model which were transplanted by human renal carcinoma 786-0 cells. PART THREE INHIBITION OF RENAL CANCER GROWTH IN NUDE MICE BY SIRNA TARGETING ON SURVIVINObjective : Human renal carcinoma 786-0 cells were used to subcutaneously inoculate BALB/C female nude mice to develop a transplanted tumor model of human renal carcinoma. The mice were treated by survivin-specific small-interfering RNA via intratumoral injection. We examined expression of survivin in tumor model of human renal carcinoma.Methods: A human renal carcinoma animal model was set up by hypodermic injection of Human renal carcinoma 786-0 cells into nude mice.When the tumor diameter reached 5~6mm, the tumor-carrying nude mice wer randomly divided into eight groups. And then, the mice were treated by survivin-specific small-interfering RNA via intratumoral injection, i.e., group A (pGenesil1.1-survivin1) and group B (pGenesil1.1-survivin1).Tumor volumes in each group were calculated every day. Three days after the last injection, all the mice were sacrificed. The tumors were taken out to weight and undergo RT-PCR and Western blot to detect the survivin expression. Tumor cell apoptosis was detected by TUNEL assay. The tumors, livers and kidneys were undergone pathologic diagnosis.Results: The tumor model of human renal carcinoma was successfully set up. The mice in groups A and B indicated slower tumor growth, significantly smaller tumor volume (P < 0.05), and lower survivin expression level (P<0.05) in comparison with those in the control groups. The TUNEL results showed a large amount of apoptotic cells with tawny nuclei, condensed chromatin and irregular cell shapes in group A and B(P<0.01). The pathologic diagnosis of the tumor is RCC.No significant pathologic abnormal was detected in the livers and kidneys in nude mice of every group.Conclusions:survivin-specific small-interfering RNA can obviously and specifically down-regulate survivin gene expression,inhibits cell proliferation and induces cell apoptosis in the transplanted tumors of human renal carcinoma in nude mice.