南方医科大学学报

南方医科大学学报 ›› 2019, Vol. 39 ›› Issue (10): 1239-.doi: 10.12122/j.issn.1673-4254.2019.10.17

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苦参碱通过调控β-catenin信号通路抑制肝癌细胞干性

戴美琴，蔡茁，陈娜娜，李金州，温嘉泳，谭丽转，郭丹   

  • 出版日期:2019-10-20 发布日期:2019-10-20

Matrine suppresses stemness of hepatocellular carcinoma cells by regulating β-catenin signaling pathway

  • Online:2019-10-20 Published:2019-10-20

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摘要： 目的探讨苦参碱对人肝癌HepG2和Huh7细胞增殖活性、细胞干性、β-catenin转录活性以及AKT/GSK3β/β-catenin信号 通路的影响。方法应用MTT法检测细胞增殖活性，平板克隆形成实验检测0 g/mL、200 g/mL、400 μg/mL、800 μg/mL苦参碱对 人肝癌HepG2 和Huh7 细胞的克隆形成能力，荧光定量PCR分析0 g/mL、200 g/mL、400 μg/mL、800 μg/mL 苦参碱对人肝癌 HepG2 和Huh7 细胞干性基因CD90、EpCAM（上皮细胞粘附分子）和CD133 mRNA的表达水平，双荧光素酶报告基因检测 0 g/mL、200 g/mL、400 μg/mL、800 μg/mL 苦参碱对人肝癌HepG2 和Huh7 细胞内β-catenin 转录活性，Western blotting 检测 0 g/mL、400 μg/mL、800 μg/mL苦参碱对人肝癌HepG2和Huh7细胞内AKT（蛋白激酶B）、GSK-3β和β-catenin及其相应磷酸化 蛋白的表达水平。结果MTT实验结果显示，苦参碱呈时间-浓度依赖性地抑制肝癌HepG2 和Huh7 细胞的增殖，处理24、 48、72 h 对HepG2 细胞的半数抑制浓度依次为2369、1565、909.1 μg/mL，对Huh7 细胞的半数抑制浓度依次为1355、781.8、 612.8 μg/mL。苦参碱浓度依赖性地抑制肝癌细胞的克隆形成能力，400 μg/mL、800 μg/mL苦参碱能够显著抑制HepG2细胞的 克隆形成能力（P<0.01，P<0.001），200 g/mL、400 μg/mL、800 μg/mL苦参碱能够显著抑制Huh7细胞的克隆形成能力（P<0.05，P<0.01， P<0.001）。苦参碱能够显著肝癌细胞内CD90、EpCAM和CD133 等干细胞标志物mRNA的表达，其中400 μg/mL、800 μg/mL 苦参碱能够显著抑制HepG2细胞中CD90（P<0.01，P<0.001）、EpCAM（P<0.05，P<0.01）和CD133（P<0.01，P<0.01）mRNA的表 达，400 μg/mL、800 μg/mL苦参碱能够显著抑制Huh7细胞中CD90（P<0.01，P<0.01）、EpCAM（P<0.05，P<0.01）和CD133（P<0.01， P<0.01）mRNA的表达。同时400 μg/mL、800 μg/mL苦参碱显著抑制HepG2细胞（P<0.001，P<0.001）和Huh7细胞（P<0.01，P<0.001） 内β-catenin的转录活性。研究结果显示400 μg/mL、800 μg/mL苦参碱能够显著降低HepG2和Huh7细胞内β-catenin和磷酸化 的AKT和GSK-3β蛋白水平，增加β-catenin的磷酸化水平。结论苦参碱可抑制肝癌HepG2和Huh7细胞的增殖、克隆形成以及 肝癌干性基因CD90、EpCAM和CD133的表达，其机制可能与抑制AKT/GSK3β/β-catenin信号通路，从而降低β-catenin的转录 活性有关。

Abstract: Objective To explore the effects of matrine on the proliferation, tumor cell stemness, β-catenin transcriptional activity and AKT/GSK3β/β-catenin signaling pathway in human hepatocellular carcinoma (HCC) HepG2 and Huh7 cells. Methods The proliferation and colony formation ability of HepG2 and Huh7 cells treated with 200, 400, and 800 μg/mL matrine were evaluated with MTT assay and colony formation assay, respectively. Real-time quantitative PCR was performed to detect the mRNA expressions of CD90, epithelial cell adhesion molecule (EpCAM) and CD133, and dual-luciferase assay was used to detect the transcriptional activity of β-catenin in the treated cells. The effects of matrine on the expressions of protein kinase B (AKT), P-AKT, GSK-3β, P-GSK-3β, P-β-catenin and β-catenin proteins in the Wnt/β-catenin signaling pathway were assessed using Western blotting. Results Matrine inhibited the proliferation of the two HCC cell lines in a time- and concentration-dependent manner. The half-inhibitory concentrations of matrine were 2369, 1565 and 909.1 μg/mL at 24, 48 and 72 h in HepG2 cells , respectively, and were 1355, 781.8, and 612.8 μg/mL in Huh7 cells, respectively. Matrine concentrationdependently suppressed colony formation of the HCC cells, producing significant inhibitory effects at 400 μg/mL (P<0.01) and 800 μg/mL (P<0.001) in HepG2 cells and at 200 μg/mL (P<0.05), 400 μg/mL (P<0.01), and 800 μg/mL (P<0.001) in Huh7 cells. Matrine at 400 and 800 μg/mL significantly inhibited the mRNA expression of CD90, EpCAM and CD133 and the transcriptional level of β-catenin in both HepG2 and Huh7 cells (P<0.05 or 0.01). Matrine at 400 and 800 μg/mL also significantly decreased the protein levels of β-catenin, P-AKT and P-GSK-3β and increased the phosphorylation level of β-catenin in both of the cell lines. Conclusion Matrine inhibits the proliferation, colony formation, and the expressions of tumor stem cell markers CD90, EpCAM and CD133 in both HepG2 and Huh7 cells probably by inhibiting Wnt/β-catenin signaling pathway and the transcriptional activity ofβ-catenin.