The In Vitro Study Of RNA Interference Targeted Survivin Gene Treated The Renal Cancer In Cell Line 786-O

RNA interference(RNAi) has recently been used to silence gene expression in various species.It has been a hot spot in studying the function of genes and the antivirus and anti-tumor therapies.As others gene therapy technique,the most difficult in using is how to choice its target gene.Survivin,a novel structurally unique inhibitor of apoptosis(IAP),is undetectable in terminally differentiated adult tissues,but becomes notably expressed in most common human cancers.Besides its role in tumor cell apoptosis and cell division,Survivin is also important in the generation of tumors blood vessels,tumor's resistance to chemotherapy and radio-therapy.Since it's found,Survivin has been acknowledged as the ideal target of gene therapy.Renal cancer is one of frequent malignancies of urinary system.It associated with the highest incidence and mortality rate among all urinary tumors.At present,the main therapy of human renal cancer is surgery, combined with chemotherapy.But,Because its mostly were discovered in late,the surgery effect was poor or had no opportunity to receive operating at all,and the serious resistant to chemotherapy drugs,renal cancer had the highest fatality rate.It is badly need to seek a new therapeutic tool.At present,it's indicated that Survivin is highly expressed in renal cancer.In this study,on the basis of Survivin's high expression in 786-O,we have transfected shRNA-Survivin with a vector of pGenesil-1 into human renal cancer cell line 786-O.ShRNA-Survivin effectively repressed the expression of Survivin in mRNA and protein level and the activity of 786-O.All of this gives us basic data of RNAi targeting Survivin in therapy of human renal cancer in vitro.Objective:PAPTⅠ- The study was designed to examine the expression of survivin gene in renal cancer cell line 786-O,then discussed its significance.PAPTⅡ:The siRNA was designed to aim directly at survivin gene and was connected with plasmid pGenesil-1,then chose the masculine coenobium and culture amplification.PAPTⅢ:The study was designed to examine the effect of shRNA-Survivin in renal cancer cell line 786-O in vitro.Methods:PAPTⅠ:We cultured the renal cancer cell line 786-O and drew its growth curve.RT-PCR was used to detected expression of survivin mRNA in the cell line,expression of survivin gene protein was measured by Western-blot.PAPTⅡ:According to survivin gene order in Genebank, we designed three masculine siRNA and a negative one.Plasmid pGenesil-1 was connected with them,and they were transformed in DH5α. The coenobium of anti-kanamycin was chosen and cultured.Finally,the plasmid of all groups was extracted.PAPTⅢ:We transfected shRNA-Survivin and control plasmid pGenesil-1 into 786-O.Cell appearance and the rate of transfection were recorded.The growth curves of every group after transfection were drawn with MTT.RT-PCR was used to detected expression of survivin mRNA in every group,expression of survivin gene protein was measured by Western-blot.Results:PAPTⅠ:Renal carcinoma cell 786-0 line was cultured with 1640 medium detected the double time is 52.2h by MTT assay.RT-PCR and western blot showed that survivin gene was highly expressed in cell line 786-O.PAPTⅡ:We got three masculine siRNA(survivinl GGACCACCGCATCTCTACA 166;survivin2 GCATTCGTCCGGTTGCGCT 358;survivin3 GGCTGGCTTCATCCACTGC 241) and one negative siRNA(GACTTCATAAGGCGCATGC).PAPTⅢ:Cell appearance of each group was similar at the 24^th hour after transfection and green fluor can be overviewed in group A,B,C,D.At the 54^th hour after transfection there were more green fluor than prophase,some cells of each group grew round and the refraction of cells cut down.There were many necrosis cells in group A,C more than group B,D,E.The transfection rate of A,B,C,D was about 35%,but E was 0.It was discovered that survivin mRNA and protein were inhibited in group S1 and S3 by RT-PCR and Western-blot. Conclusions:PAPTⅠ:Survivin gene was over-expressed in renal cancer cell line786-O,which suggested that survivin gene may play an important role in the development of renal cancer.PAPTⅡ:The siRNA which designed to aim directly at survivin gene in vitro offered a valuable arm for us to restrain expression of survivin in 786-O.PAPTⅢ:Sequence specific siRNA targeting survivin can efficiently inhibit the survivin expression and cell proliferation in renal cancer cell line 786-O.The successful application of survivin extends the list of available therapeutic modalities in the treatment of renal cancer.