miRNA通过Mcl-1基因调控HBV阳性肝癌细胞对索拉菲尼的敏感性

miRNA regulating the sensitivity of HBV-positive hepatocellular carcinoma cells to sorafenib by Mcl-1 gene

目的:探讨干预HBV阳性肝癌细胞相关miRNAs对肝癌细胞索拉菲尼敏感性的影响。方法:qPCR检测HepG2.2.15(HBV阳性)和HepG2.vc(HBV阴性)肝癌细胞中miR-29、miR-101和miR-193b的表达,以HepG2.2.15细胞中低表达的miRNA合成相应的miRNA mimics,并将目标miRNA mimics分别转染至HepG2.2.15和HepG2.vc细胞;qPCR检测两种细胞中目标miRNA表达,Western blotting检测目标miRNA mimics转染前后Mcl-1蛋白表达。同时分别向转染和非转染的HepG2.2.15和HepG2.vc细胞中分别加入(1×10^(-9))^(1×10^(-3))mol/L的索拉菲尼,72 h后测定索加菲尼对细胞作用的IC_(50)值和细胞凋亡率。结果:与HepG2.vc细胞比较,HepG2.2.15细胞中miR-193b的表达显著降低(P<0.05);与miR-193b mimics转染前比较,HepG2.2.15和HepG2.vc细胞中miR-193b的表达均有显著升高(P<0.05)。与HepG2.vc细胞比较,HepG2.2.15细胞中Mcl-1蛋白表达显著增高(P<0.05);miR-193b mimics转染后,HepG2.2.15和HepG2.vc细胞中Mcl-1蛋白表达较两者转染前均有显著降低(P<0.05);miR-193b mimics转染后,索拉菲尼可显著增加两组细胞的凋亡率(P<0.01),同时其对两组细胞作用的IC_(50)值显著降低[HepG2.2.15细胞:(0.215±0.028)vs(0.391±0.025)mol/L,HepG2.vc细胞:(0.315±0.027)vs(0.654±0.019)mol/L;均P<0.01]。结论:HBV相关肝癌细胞中miR-193b的低表达可能是癌细胞对索拉菲尼敏感性降低的原因,Mcl-1可能为miR-193b的靶点,miR-193b mimics与索拉菲尼具有显著协同作用。

Objective： To investigate the miRNAs that can intervene Mcl-1 expression in HBV-related liver cancers and to study their synergistic anti-cancer effect with sorafenib. Methods： The expressions of miR-29, miR-101 and miR-193b in HepG2.2.15（HBV positive） and HepG2.vc（HBV negative） cells were detected by qPCR. miRNA mimics of low expressed genes in HepG2.2.15 cells were synthesized and transfected into HepG2.2.15 and HepG2.vc cells, respectively. qPCR was used to detect target miRNA expression.Western blotting was used to detect the expression of mcl-1 protein in cells before and after transfection. At the same time,（1×10^（-9））^（1×10^（-3）） mol/L of sorafenib was add to both transfected and non-transfected HepG2.2.15 and HepG2.vc cells; 72 h later, the IC_（50） and cell apoptosis was evaluated. Results： The expression of miR-193b in HepG2.2.15 cells was significantly lower than that in HepG2.2.15 cells（P 0.05）. The expression of miR-193b in HepG2.2.15 cells and HepG2.2.15 cells was significantly higher after miR-193b mimics transfection（P 0.05）. Compared with HepG2.vc cells, the expression of Mcl-1 protein in HepG2.2.15 cells was significantly increased（P 0.05）. The expression of Mcl-1 protein in HepG2.2.15 and HepG2.vc cells was significantly decreased after miR-193b mimics transfection（P0.05）. After miR-193b mimics transfection, sorafenib could significantly increase apoptosis rate of both HepG2.2.15 and HepG2.vc cells. Conclusion： The low susceptibility of HBV-related liver cancer to sorafenib may be related with the low expression of miR-193b in cancer cells. Mcl-1 might be used as a target of miR-193b, and miR-193b mimics have a significant synergistic effect with sorafenib.