RNA干扰介导的MDR1和MRP1基因沉默逆转肝细胞癌多药耐药

Reversal of multidrug resistance of HepG2 by RNAi-mediated gene silence of MDR1 and MRP1

目的 探讨RNAi技术体外联合抑制MDR1和MRP1基因逆转肝细胞癌多药耐药的效果.方法 建立肝细胞癌多药耐药细胞株,分别构建MDR1和MRP1基因的shRNA的质粒载体,转染肝细胞癌多药耐药细胞株.设空白对照组、MDR1单基因抑制HepG2/MDR-1-si组、单基因抑制HepG2/MRP-1-si组、联合基因抑制HepG2/mm-si组.流式细胞术检测细胞周期及凋亡,MTT法检测转染后细胞耐药性,CCK-8试剂盒检测细胞活性及相对增值情况,caspase-3活性检测用分光光度仪,实时PCR测定MDR1/mRNA、MRP1/mRNA.结果 成功构建pSUPER-HepG2/MDR-1-si、pSUPER-HepG2/MRP-1-si.MRP-1-mRNA在HepG2/mm-si组与针对该基因设计的单基因沉默组（HepG2/MRP-1-si）的表达差异无统计学意义（P＞0.05）;在HepG2/mm-si组与HepG2/MDR-1-si）组的表达差异亦无统计学意义（P＞0.05）.HepG2/mm-si组Caspase-3活性明显高于HepG2/MRP-1-si,差异有统计学意义（14 623.7±338.9比13 215.7 ±90.6,P ＜0.05）.CCK-8法检测显示双基因沉默组的细胞活性、细胞数目与其他各组比较明显降低,差异有统计学意义（P＜0.05）.对照组HepG2/MDR-1-si、HepG2/MRP-1-si的耐药性分别为HepG2/mm-si组的1.39倍和1.58倍.HepG2/mm-si组耐药细胞株凋亡期细胞数量明显少于HepG2/MRP-1-si和HepG2/MDR-1-si组,差异有统计学意义（P＜0.01）.结论 siRNA体外联合抑制MDR1和MRP1基因可以显著逆转肝细胞癌多药耐药.

Objective To investigate the in vitro effect of silencing of gene MDR1 and MRP1 by siRNA on multidrug resistance reversal in hepatocellular carcinoma.Methods Plasmid vector of shRNA for gene MDR1 and MRP1 were constructed to transfect the multidrug resistance of hepatocellular carcinoma cell lines.Cells were grouped into control （HepG2）,single gene silent （HepG2/MDR-1-si,HepG2/MRP-1-si）,and combined gene silent （HepG2/mm-si）.Cell cycle and apoptosis were measured by flowcytometry,and the resistance of transfected cells were measured by MTT.Cell activity and cell proliferation can be detected by CCK-8,activity of caspase-3 by A450,MDR1/mRNA and MRP1/mRNA by real-time PCR.Results Cell lines of pSUPER-HepG2/MDR-1-si and pSUPER-HepG2/MRP-1-si were constructed successfully.MRP-1-mRNA of HepG2/mm-si was equal to that of HepG2/MRP-1-si,and MDR-1-mRNA of HepG2/mm-si equals to that of HepG2/MRP-1-si （P ＞ 0.05）.Activity of caspase-3 in HepG2/mm-si was higher than that of HepG2/MRP-1-si （14 623.7 ± 338.9 vs.13 215.7 ± 90.6,P ＜ 0.05）.Cell activity and cell proliferation in HepG2/mm-si were lower than that of other groups （P ＜ 0.05）.The sensitivity of HepG2/MDR-1-si and HepG2/MRP-1-si was higher than that of HepG2/mm-si （1.39-fold vs.1.0-fold,1.58-fold vs.1.0-fold,P ＜ 0.05）.Cell count of apoptosis in HepG2/MDR-1-si and HepG2/MRP-1-si was larger than that of HepG2/mm-si （P ＜ 0.01）.Conclusion In vitro silence of MDR1 and MRP1 gene by RNA interference can reverse the multidrug resistance of HepG2 cell line.