Impact Of RNA Interference ERCC1 Gene Expression On DDP And PGPIPN Sensitivity Of Ovarian Cancer Drug Resistance Cell

Abstract Objective To design and construct the expression vector of human ERCC1 gene shRNA lentivirus plasmid, and establish the stably transfected cell strain of human ovarian cancer resistant cell SKOV3/DDP to explore the new methods to treat ovarian cancer resistance.MethodAccording to the sequence of human ERCC1 gene, design 3 siRNA sequences for ERCC1 gene to transiently transfect human ovarian cancer resistant cell SKOV3/DDP, and after selection of the most effective silencing target gene siRNA, design and synthesize shRNA sequences being able to express its small hairpin structure, connect and transform with lentiviral vector plasmid pLVX-shRNA1 of double enzyme digestion via BamH I and EcoR I, pick single colony after screening, extract plasmid after PCR and sequencing identification, lentiviral recombinant plasmid lentiviral pLVX-shRNA1-ERCCl and lentiviral packaging auxiliary plasmid psPAX2 and pMD2.G co-transfect packaging cell 293T cell, collect virus supernatant, measure viral titers, transfect SKOV-3/DDP cells, use puromycin to screen stably transfected pLVX-shRNAl-ERCC1 of SKOV-3 / DDP cell strain, and finally use RT-PCR identification interference effect.ResultPCR and sequencing identification results proved that double-stranded shRNA successfully inserted into lentiviral vector pLVX-shRNAl, co-transfected 293T cell obtained high titer virus with a suspension titer of108IFU, SKOV-3/DDP cell strain of stably transfected pLVX-shRNAl-ERCC1 was obtained after SKOV3/DDP was transfected, and ERCC1 mRNA gene expression detected in PCR identification declined significantly.Conclusion application of RNA interference technology, efficient and specific expression of silencing ERCC1 gene, RNAi technology for ovarian cancer ERCC1, could become a new clinical method to treat ovarian cancer.Abstract Objective Discuss the impact of RNA interference ERCC1 gene express ion on DDP and PGPIPN sensitivity of ovarian cancer drug resistance cell.Methods Design and Compound the RNA interference double-stranded DNA in v itro, transfect the human ovarian cancer drug resistance cell SKOV3/DDP, and es tablish stable SKOV3/DDP cell line of pLVX-shRNA-ERCC1 stable transfection as well as SKOV3/DDP cell line of pLVX-shRNA-Negative control stable tran sfection. The experiment is divided into the following 3 groups:blank control gr oup (human ovarian cancer drug resistance cell line SKOV3/DDP cell), specificit y transfection group (SKOV3/DDP cell of pLVX-shRNA-ERCC1 stable transfectio n) and non-specificity transfection group (SKOV3/DDP cell of pLVX-shRNA-Neg ative control stable transfection). MTT method was used to determine the effects of PGPIPN, DDP on cell proliferation inhibition rate for the three groups; RT-P CR method was used to test the effects of PGPIPN on gene expression of breast cancer susceptibility gene 1(BRCA1) in cell of the three groupsce cell for 48h,a nd provided certain inhibition for their proliferation, which was statistically sign. Western blot method was used to test the effects of PGPIPN on BRCA1 protein expression in cell of the three groupsce cell for 48 h.Results MTT results showed that PGPIPN affected human ovarian cancer drug re sistan ificant at 40,60 mg/L of concentration compared to 0 mg/L(P<0.05); DDP and PGPIPN affected cell for 48 h respectively in specificity transfection group, in which the cell proliferation significantly was inhibited, both of which have sta tistically significant differences compared to 0 mg/L of DDP.PGPIPN concentratio n(P<0.05); RT-PCR results showed that PGPIPN affected cells for 48 h in specifi city transfection group, BRCA1 gene expression of which significantly decreased with the increase of PGPIPN concentration, having statistically significant differen ce at 40,60 mg/L of concentration compared to 0 mg/L of PGPIPN concentratio n(P<0.01). Western blot results showed that PGPIPN affected cells for 48 h in s pecificity transfection group, BRCA1 protein expression of which significantly de creased with the increase of PGPIPN concentration, having statistically significant difference at 60 mg/L of concentration compared to 0 mg/L of PGPIPN concent ration(P＜0.01).Conclusion PGPIPN have certain inhabitation effect on the proliferation of SKO V3/DDP, and it can significantly enhance the DDP and PGPIPN sensitivity by in terference ERCC1 gene expression of SKOV3/DDP. Meanwhile, it can enhance P GPIPN down-regulate the level of BRCA1 gene and protien expression.