短发夹RNA干扰表达质粒逆转人大肠癌细胞LOVO/5-Fu多药耐药的研究

Reversal of multidrug resistance in human colon carcinoma LOVO/5-Fu cells by short hairpin RNA expression plasmids

目的:探讨短发夹RNA(shRNA)沉默多药耐药MDR)基因对大肠癌多药耐药细胞株LO-VO/5-Fu生长的影响。方法:构建靶向MDR1的shRNA干扰质粒转染人大肠癌多药耐药细胞株LO-VO/5-Fu,MTT法检测各组细胞对5-Fu的敏感性,流式细胞术检测细胞周期变化、凋亡情况及P-糖蛋白(P-gp)的表达,Realtime-PCR检测各组细胞MDR1 mRNA表达的变化,Western blot检测各组细胞P-gp表达的变化。结果:与对照组质粒组和未转染组相比,转染含shRNA干扰质粒细胞的实验组IC50明显降低,为(2.304±0.232)μmol/L,且差异有统计学意义(P<0.05),敏感性的相对逆转率为73.8%;凋亡率明显升高为(5.767±0.694)%(P<0.05);MDR1 mRNA表达明显下调(P<0.05);P-gp的表达水平降低(P<0.05)。结论:靶向MDR1的shRNA干扰质粒有效抑制了MDR1的表达,使P-gp的表达降低,从而增强了LOVO/5-Fu细胞对5-Fu的敏感性,有可能为临床上克服大肠癌化疗过程中出现的耐药提供新的作用靶点和治疗途径。

Objective： To explore the effect of MDR1 gene silence on the cell viability of human colon carcinoma cell line LOVO/5-Fu by short hairpin RNA.Methods： A eukaryotic expression plasmid of shRNA targeting on MDR1 was constructed and was transiently transfected into human colon carcinoma LOVO/5-Fu cells.Drug sensitivity was measure by MTT.The Cell cycle,apoptosis of cells and expression of P-glycoprotein（P-gp） were determined by flow cytometry assay.Expression of MDR1 mRNA was detected by Real-time PCR.P-gp expression was detected by using Western blot.Results： The IC50 of 5-Fu in MDR1 shRNA-transfected group was reduced by（2.304±0.232） μmol/L as compared with that in negative control and empty vetor-transfected group（P〈0.05）,the relative reverse rate of sensitivity of LOVO/5-Fu cells to 5-Fu was 73.8%;and the apoptotic rate increased to（5.767±0.694）%（P〈0.05）;the expression of MDR1 mRNA and P-gp were reduced obviously（P〈0.05）.Conclusions： The eukaryotic expression plasmid of shRNA targeting on MDR1 effectively inhibites the expression of MDR1,reduces the expression of P-gp,thus enhances the sensitivity of LOVO/5-Fu cells to 5-fluorouracil.So MDR1 gene silence may provide a new target and an effective way against drug resistance of 5-fluorouracil.