RNA干扰逆转人胃癌细胞多药耐药的研究

Reversal of multidrug resistance in human gastric carcinoma cells by RNAi

目的:研究RNA干扰沉默多药耐药(multidrug resistance,MDR)基因对胃癌多药耐药细胞株BGC-823/5-Fu生长的影响。方法:构建靶向MDR1的shRNA干扰质粒转染人胃癌多药耐药细胞株BGC-823/5-FU,噻唑蓝(MTT)法检测耐药细胞对5-Fu的敏感性,实时荧光定量PCR(Real-time-PCR)检测MDR1 mRNA表达的变化,Western blot检测各组细胞P-gp表达的变化,流式细胞术检测细胞周期变化、凋亡情况。结果:与RNAi-control组和normal组相比,RNAi-MDR1组细胞干扰质粒细胞的IC50明显降低,为2.104±0.242(P<0.05),敏感性的相对逆转率为76.8%;MDR1 mRNA表达明显下调(P<0.05);P-gp的表达水平降低(P<0.05);凋亡率明显升高至(5.757±0.684)%。结论:应用RNA干扰有效抑制了MDR1的表达,使P-gp的表达降低,增强了BGC-823/5-Fu细胞对5-Fu的敏感性,为寻找逆转胃癌细胞多药耐药有效方法奠定了基础。

Objective： To explore the effect of MDR1 gene silence on the cell viability of human gastric carcinoma cell line BGC-823/5-Fu by RNAi.Methods： A eukaryotic expression plasmid of shRNA targeting on MDR1 was constructed and was transiently transfected into human gastric carcinoma BGC-823/5-Fu cells.Drug sensitivity was measure by MTT.Expression of MDR1 mRNA was detected by real-time quantitative PCR（Real-time PCR）.P-gp expression was detected by using Western blot.The Cell cycle and apoptosis of cells were determined by flow cytometry assay.Results： The IC50 of 5-Fu in MDR1 shRNA-transfected group was reduced by 2.104±0.242（P〈0.05）as compared with that in negative control and empty vetor-transfected group,the relative reverse rate of sensitivity of BGC-823/5-Fu cells to 5-Fu was 76.8%;;the expression of MDR1 mRNA and P-gp were reduced obviously（P〈0.05） and the apoptotic rate increased to（5.757±0.684）%（P〈0.05）.Conclusion： The RNAi targeting on MDR1 effectively inhibited the expression of MDR1,reduced the expression of P-gp,thus enhance the sensitivity of BGC-823/5-Fu cells to 5-fluorouracil.