Study Of RNA Interference Suppression ERCC2Gene Expression In Human Lung Adenocarcinoma Resistant Cells

Objective:Explore the RNA interference excision repair cross complementing group2in the human lung adenocarcinoma resistant cells A549/DDP of expression, to provide ex-perimental evidence for Non-small cell lung cancer gene therapy.Methods:ERCC2gene in vitro design and synthesis of targeted people, including small interfering RNA (siRNA):ERCC2-siRNA1, ERCC2-siRNA2, ERCC2-siRNA3, ERCC2-siRNA4and negative control group (NC ERCC2-siRNA) construct carrying of ERCC2-the shRNA plasmid expression vector, cationic polymer EndoFectinTM Lenti transfected into human lung adenocarcinoma drug-resistant cell lines A549/DDP observed using fluorescence microscopy and determination of transfection efficiency; application of real-time PCR, detect transfected ERCC2mRNA to expression.Results:1. A549/DDP48h after cell transfection observed under a fluorescence microscope to each transfection group have different proportions of eGFP expression, blank control group, no eGFP expression, the transfection efficiency of the best of ERCC2-siRNA2(83.45±3.27%).2. The expression of ERCC2mRNA in A549/DDP cells after transfection for24,36,48hours was detected by real-time PCR. After transfection for24hours, The ERCC2mRNA relative expression value of ERCC2-siRNA1group, ERCC2-siRNA2group, ERCC2-siRNA3group and ERCC2-siRNA4group were0.821±0.112,0.452±0.087,0.591±0.127,0.785±0.135(P<0.05).respectively and compared with blank control and NC ERCC2-siRNA group, the ERCC2mRNA expressions level in A549/DDP cells were all down-regulated (P<0.05), the most obviously of them was ERCC2-siRNA2(P<0.01); After transfection for36hours, The ERCC2mRNA relative expression value of ERCC2-siRNA1group, ERCC2-siRNA2group, ERCC2-siRNA3group and ERCC2-siRNA4group were0.818±0.101、0.358±0.097、0.576±0.081、0.772±0.174(P<0.05).respectively and compared with blank control and NC ERCC2-siRNA group, the ERCC2mRNA expressions level in A549/DDP cells were all down-regulated (P<0.05), the most obviously of them was ERCC2-siRNA2(P<0.01); After transfection for48hours, The ERCC2mRNA relative expression value of ERCC2-siRNA1group, ERCC2-siRNA2group, ERCC2-siRNA3group and ERCC2-siRNA4group were0.813±0.183,0.293±0.118,0.564±0.086,0.763±0.078(P<0.05) respectively and compared with blank control and NC ERCC2-siRNA group, the ERCC2mRNA expressions level in A549/DDP cells were all down-regulated (P<0.05), the most obviously of them was ERCC2-siRNA2(P<0.01).Conclusion:1In this study, the design and synthesis of plasmid expression vector can be cationic polymerization EndoFectinTM the Lenti successfully transferred to drug-resistant cell lines of human lung adenocarcinoma A549/DDP.2RNA interference technology may be some downward adjustment in human lung adenocarcinoma resistant cell A549/DDP ERCC2mRNA gene expression levels.3Construction of the target sequence of ERCC2-siRNA2interence silencing effect is best.