小干扰RNA逆转Wnt/β-catenin通路对肝癌耐药细胞株HepG2/ADM的影响研究  

Effect of small interfering RNA reverse wnt/β-catenin pathway on hepatocellular carcinoma cell line Hep G2/ADM

作　　者：张华耀 谭浪平[2] 刘建平[2] 苏正[2] 韦金星[2] 黄延年 ZHANG Huayao;TAN Langping;LIU Jianping;SU Zheng;WEI Jinxing;HUANG Yannian(General Surgery,Zengcheng People’S Hospital of Guangzhou,Guangzhou 511300,China;Hepatobiliary Surgery,Sun Yat-Sen Memorial Hospital of Sun Yat-sen University,Guangzhou 510120,China)

机构地区：[1]广州市增城区人民医院普通外科,广东广州511300 [2]中山大学孙逸仙纪念医院肝胆外科,广东广州510120

出　　处：《岭南现代临床外科》2019年第5期520-524,共5页Lingnan Modern Clinics in Surgery

基　　金：国家自然科学基金青年科学基金项目(81702406)

摘　　要：目的了解沉默β-catenin基因对肝癌耐药细胞HepG2的影响。方法实验分为5组:正常肝细胞(LO2)组、HepG2组、HepG2/ADM组、SiNC-HepG2/ADM阴性转染组和Siβ-cateninHepG2/ADM转染组;细胞免疫荧光技术检测各组β-catenin的表达情况;设计并筛选出抑制效率最高的Siβ-catenin;Western-blot及RT-PCR技术检测各组β-catenin、P-gp、MRP1的mRNA和蛋白的表达水平;MTT法观察各组对阿霉素(ADM)、氟尿嘧啶(5-FU)、环磷腺苷(VCR)和奥沙利铂(OHP)的敏感性;流式细胞仪检测各组细胞凋亡。结果 50 mol/L的ctnnb1-001在HepG2/ADM中抑制效率最高:78.86%(P<0.05),选其为Si-β-catenin;细胞免疫荧光显示HepG2/ADM中β-catenin荧光最强,转染Si-β-catenin后荧光显著减弱;β-catenin、P-gp和MRP1 mRNA和蛋白在HepG2/ADM组表达较高,mRNA表达分别为:0.92±0.03、7.98±0.43和4.56±0.12(P<0.05),蛋白表达分别为:1.128±0.214、1.678±0.344和1.405±0.212(P<0.05);转染Siβ-catenin至HepG2/ADM中,β-catenin、P-gp和MRP1在mRNA及蛋白水平均不同程度表达减少,mRNA表达分别为:0.47±0.03、0.66±0.054和0.74±0.03(P<0.05),蛋白表达分别为:0.787±0.032、0.797±0.055和1.390±0.050(P<0.05);Siβ-catenin-HepG2/ADM转染组较HepG2/ADM组对ADM、5-FU、VCR和OHP的耐药系数(RI)分别为0.61、0.55、0.30、0.55,对化疗药物敏感性显著增强(P<0.05);Siβ-catenin-HepG2/ADM转染组凋亡率(28.05±0.35)%,较其他组明显增加(P<0.05)。结论 Wnt/β-catenin通路在HepG2中异常激活,其中β-catenin可能正性调控肝癌耐药基因P-gp和MRP1,Si-β-catenin能一定程度阻断Wnt通路,并能一定程度逆转肝癌细胞株HepG2的耐药性和增强化疗敏感性,增加凋亡。Objective To investigate the effect of silencing β-catenin gene on HepG2 cells in liver cancer cells. Methods The experiment was divided into 5 groups:normal liver cell(LO2)group,HepG2 group,HepG2/ADM group,SiNC-HepG2/ADM negative transfection group and Siβ-catenin-HepG2/ADM transfection group;The expression of β-catenin was determined;the highest inhibition rate of Siβ-catenin was designed and screened;the mRNA and protein expression levels of β-catenin,P-gp and MRP1 were detected by Western-blot and RT-PCR techniques;MTT assay The sensitivity of each group to doxorubicin(ADM),fluorouracil(5-FU),cyclic adenosine monophosphate(VCR) and oxaliplatin(OHP)was observed. The apoptosis of each group was detected by flow cytometry. Results50 mol/L ctnnb1-001 had the highest inhibition efficiency in HepG2/ADM:78.86%(P<0.05),which was selected as Si-β-catenin. Cellular immunofluorescence showed that β-catenin had the strongest fluorescence in HepG2/ADM. After transfection of Si-β-catenin,the fluorescence was significantly attenuated;β-catenin,P-gp and MRP1 mRNA and protein were highly expressed in HepG2/ADM group,and the mRNA expressions were 0.92±0.03,7.98±0.43 and 4.56±0.12,respectively. P<0.05),protein expression was 1.128±0.214,1.678±0.344 and 1.405±0.212(P<0.05);transfection of Siβ-catenin to HepG2/ADM,β-catenin,P-gp and MRP1 in mRNA and The expression levels of protein were decreased at different degrees. The mRNA expressions were 0.47±0.03,0.66± 0.054 and 0.74± 0.03,respectively(P<0.05). The protein expressions were 0.787 ± 0.032,0.797 ± 0.055 and 1.390 ± 0.050,respectively(P<0.05). The resistance coefficient(RI)of Aβ,5-FU,VCR and OHP in the Siβ-cateninHepG2/ADM transfection group was 0.61,0.55,0.30 and 0.55,respectively,compared with HepG2/ADM group,and the sensitivity to chemotherapy drugs was significant. The enhancement rate was(P<0.05). The apoptosis rate of Siβ-catenin-HepG2/ADM transfection group was 28.05±0.35%,which was significantly higher than other groups(P<0.05). Conclusion Th

关 键 词：肝癌 多药耐药 β-catenin 小干扰RNA HEPG2 

分 类 号：R735.7[医药卫生—肿瘤]