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

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。

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

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.