Study Of RNAi Targeting Epidermal Growth Factor Receptor On Lung Adenocarcinoma A549 Cell Line Growth And Sensitivity To Cisplatin

Objective:Using RNA interference(RNAi) technique,to observe the effect of recombinant plasmid carrying epidermal growth factor receptor(EGFR) homologous gene on the growth-inhibiting effect of A549 cell and sensitivity to cisplatin after transfected to non-small cell lung cancer A549 cell.Method: According to the principle of RNAi, a strip of interferencing fragment targeting EGFR gene ,h-EGFR ,was designed and synthesized, then pGensil-1 was used as vector, the recombinant plasmid pGensil-1-EGFR(ds-EGFR)was constructed and transfected into human lung cancer A549 cell line mediated by Lipofectamine 2000.Then the transfected cells were selected with G418 and the stable transfected monoclone cells were picked out .After collected and cultured,cells were treated with different concentrations of cisplatin(2.5ug/ml,10ug/ml,40ug/ml) for different time (24h,48h,72h).The antiproliferative efects of dsRNA and cisplatin were assessed by MTT.Cell cycle analysis and the apoptosis rates were carried out via flow cytometry. Result: From the second day,ds-EGFR group grew slower, the optical density value were 0.741±0.010,0.860±0.054,0.999±0.192 at 48h,72h,96h,down-regulated significantly compared with control group(P<0.05).ds-EGFR combined with different concentrations(2.5ug/ml,10ug/ml,40ug/ml )of cisplatin for 24h,the inhibition ratio increased by 7.8%,30.7%,34.42%,compared with isoconcentration cisplatin.This indicated ds-EGFR combined with cisplatin or cisplatin can inhibit cell grow and this effects of cisplatin were in a dose-time-dependent manner.Cell cycle analysis showed that ds-EGFR induced accumulation of cells in G0-G 1 phase by 13.84% with a significant decrease in the percentage of cells in S-phase by 1 9.10% relative to the contro1.After 24hours treated with cisplatin of 40ug/l ,10ug/l ,2.5ug/l,the apoptosis rates increased compared with control group(P<0.05).After treated with cisplatin of 40ug/l ,10ug/l ,2.5ug/l for 24h, apoptosis rates were 34.40±1.21,16.30±1.19,7.22±1.25, and after treated with ds-EGFR combined with cisplatin of 40ug/l ,10ug/l ,2.5ug/l, apoptosis rates were37.12±1.99,17.93±1.13 ,10.42±1.45. Conclusion:ds-EGFR could effectively inhibit cell growth, make more cells block in the G0-G1 phase and increse the sensitivity of A549 cell to cisplatin .This provides a experimental basis for RNAi technique in gene therapy on NSCLC and indicates that RNAi combined with cisplatin have synergistic effect.