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Lingnan Modern Clinics in Surgery ›› 2019, Vol. 19 ›› Issue (05): 520-524.DOI: 10.3969/j.issn.1009-976X.2019.05.002

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

ZHANG Huayao1, TAN Langping2, LIU Jianping2, SU Zheng2, WEI Jinxing2, HUANG Yannian1.   

  1. 1. General Surgery, Zengcheng People’S Hospital of Guangzhou, Guangzhou 511300, China; 2. Hepatobiliary Surgery, Sun Yat-Sen Memorial Hospital of Sun Yat-sen University, Guangzhou 510120, China
  • Contact: Huang Yannian

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

张华耀1, 谭浪平2, 刘建平2, 苏正2, 韦金星2, 黄延年1*   

  1. 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
  • 通讯作者: 黄延年

Abstract: 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. Results 50 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-catenin-HepG2/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 The Wnt/β-catenin pathway is aberrantly activated in HepG2. β-catenin may positively regulate the drug resistance genes P-gp and MRP1. Si-β-catenin can block the Wnt pathway to a certain extent and reverse liver cancer to some extent. The drug resistance of the cell line HepG2 enhances chemosensitivity and increases apoptosis.

Key words: β-catenin, HepG2, liver cancer, multidrug resistance, small interfering RNA

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-catenin-HepG2/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。

关键词: 肝癌, 多药耐药, β-catenin, HepG2, 小干扰RNA

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