shRNA介导的RNAi载体构建及对肝癌耐药细胞MDR1基因表达的抑制

shRNA-mediated construction of RNAi expression plasmid and its inhibitory effect on MDR1 expression in hepatocellular carcinoma cell SMMC-7721/R

目的:重组构建抑制多药耐药MDR1基因表达的shRNA真核表达载体,研究其对肝癌耐药细胞MDR1基因表达的抑制作用,以及对P-糖蛋白(P-gp)表达和功能的抑制作用.方法:根据MDR1基因序列,设计两段21个碱基的MDR1特异性靶序列作为shRNA目标序列(sh-MDR1-1,sh-MDR1-2),重组构建psh-MDR1表达质粒.采用脂质体介导转染肝癌耐药细胞SMMC-7721/R,用MTT法测定细胞对化疗药物阿霉素(ADM)的敏感性,流式细胞仪检测细胞膜表面P-糖蛋白(P-gp)表达和细胞内Rhdaming123(Rh123)的潴留.结果:PCR和DNA测序证实了psh-MDR1-1和psh-MDR1-2重组质粒的成功构建,均能有效地抑制细胞P-gp表达;转染细胞后均能够一定程度地恢复耐药细胞对化疗药物ADM敏感性,P-gp表达水平明显降低,细胞内的Rh123稳态积累量均明显增高;第1条序列更能有效的抑制MDR1基因表达.结论:体外完成shRNA RNAi载体的构建,能有效地逆转肝癌耐药细胞MDR1基因过度表达,抑制P-gp介导的多药耐药.从DNA载体产生的shRNA在肝癌耐药细胞内能诱导RNAi,能够序列特异性地抑制MDR1基因表达.

AIM ： To construct a recombinant plasmid generating short hairpin RNA（shRNA） containing multi-drug resistance gene MDR1 segment in mammalian cells and to investigate its suppression on MDR1 mRNA and P-glycoprotein （P-gp） expressions in hepatocellular carcinoma cells. METHODS ： Based on the design of two fragments of oligonucleotides for shRNA expression which targeted MDR1 gene （sh-MDRI-1, sh-MDR1-2）, RNAi plasmids （psh-MDR1）were constructed and transfected into SMMC-7721/ R cells by LyoVecTM. Drug sensitivity was measured by MTT assay, P-gp expression and intracellular Rh123 accumulation were determined by flow cytometry （FCM）. RESULTS ： Recombinant psh-MDR1 vectors were identified by PCR and confirmed by sequencing analysis. They could suppress P-gp expression in hepatocellular carcinoma cells. Drug sensitivity was increased significantly after pshRNA-MDR1 was transfected in SMMC-7721/R cells. The level of P-gp expression was reduced significantly. The intracellular accumulation of Rh123 was increased greatly after psh-MDR1 treatment in SMMC-7721/R cells. The sh-MDRI-1 was more effective in the suppression of MDR1. CONCLUSION： Recombinant psh-MDR1 vector can suppress the expressions of MDR1 mRNA and P-gp in SMMC-7721/R cells. The inhibitory effect of the shRNA generated from the DNA vector is highly related to the target sites and to the different cell lines.