Effect Of Smac Transfection On Epithelial Ovarian Carcinoma Cell Cisplatin-Resistance

Objective:1.To observe the differential expression of Smac in ovarian carcinoma cell lines of SKOV3 and SKOV3/DDP.2.An eukaryotic expressing vector pcDNA3.1(+)/Smac was constructed and trasfected into epithelial ovarian carcinoma SKOV3/DDP cell line.3.Observe the effect of transfer exogenous Smac gene on epithelial ovarian carcinoma drug-resistance cell.4.Discusses the relationship of Smac and the drug resistance of ovarian carcinoma.Method:1.Check IC50 of the drug-resistant epithelial ovarian cancer cell SKOV3/DDP to SKOV3 through MTT,and calculate resistant index.2.To observe the expression of Smac in ovarian carcinoma cell lines of SKOV3 and SKOV3/DDP by RT-PCR and western blot.3.Two-step RT-PCR was performed to get mature Smac cDNA.After the double digestion to Smac cDNA and the mammalian expression vector pcDNA3.1(+) with XhoI/BamHI,two purified target fragments were ligated by T4 DNA ligase.Then the ligation products were transformed into XL1-blue competent cells.Recombinant vector was amplified and examined by sequencing.4.Transfer pcDNA3.1(+)/Smac to SKOV3/DDP through liposome infection protocol.Test Smac expression after transfection through Western blot.And after treated with cisplatin,the invitro cellular growth activitise were estimated by MTT colorimetry.Observe the apoptosis of drug-resistant cell after transfection of Smac through Annexin-v.Results:1.The resistant index of SKOV3/DDP is 3.96,which could be used in the test.2.Smac was detected in ovarian cell lines SKOV3 and SKOV3/DDP,the relative content of Smac/DIABLO mRNA in SKOV3 was higher than that in SKOV3/DDP.3.Eukaryotic express vector pcDNA3.1(+)/Smac was constructed successfully and examined by sequencing. 4.Smac protein expression is positive after transfection through Western blot.After adding cisplatin of different concentration and different times,cell increase inhibition ratio is proved to be improved in transfecting resistant cell by MTT(P＜0.05).Apoptosis is increase after transfecting Smac to resistant cell SKOV3/DDP through Annexin-v(P＜0.05).Conclusion:Smac protein expression is lower in ovarian cell lines.Overexpression of Smac was definitely related to the rise of ovarian cancer.Upregulating the expression of Smac domains in epithelial ovarian carcinoma cell lines in vitro could weakened the tolerance of cancer cells to cisplatin.It also suggested that Smac might be involved in Cisplain-resistance of ovarian cancer cells.