目的 探索南蛇藤提取物（COE）靶向哺乳动物雷帕霉素靶蛋白（mTOR）抑制人肝癌HepG2细胞增殖、侵袭与转移的分子机制。方法 用siRNA技术，构建敲除mTOR基因的HepG2细胞模型，MTT法检测COE对HepG2/mTOR^-细胞增殖能力的影响。划痕实验及Transwell实验检测药物对细胞侵袭转移能力的影响。Western blotting法分析用药后基质金属蛋白酶-2（MMP-2）、MMP-9蛋白的表达水平变化。结果 成功构建敲除mTOR表达的HepG2/mTOR^-细胞。COE明显抑制HepG2/mTOR^-细胞的增殖（P<0.05），并呈浓度依赖性。划痕实验结果显示COE降低了细胞迁移能力。Transwell实验结果表明，COE（80 mg/L）明显减少了穿膜细胞数目（P<0.05）。Western blotting结果显示，COE（80 mg/L）明显降低MMP-2、MMP-9蛋白的表达水平（P<0.05）。结论 COE明显抑制HepG2/mTOR^-细胞的增殖与侵袭转移，mTOR通路可能是其抑制肝癌的潜在作用靶点之一。

Objective To investigate the molecular mechanisms of Celastrus orbiculatus extracts (COE) of the invasion and metastasis inhibition in human hepatocellular carcinoma HepG2 cells by targeting mTOR. Methods The HepG2/mTOR^- cells with mTOR knockout expression were constructed by using siRNA technology. The effect of COE on the proliferation of the HepG2/mTOR^-cells was also studied. The HepG2/mTOR^- cells were treated with COE in different concentrations (20, 40, 80, 160, and 320 mg/L) for 24 h. The cell reproductive capability of HepG2/mTOR^- cells was detected by MTT. The effect of COE on the metastatic ability of HepG2/mTOR^- cells in vitro was investigated by scratch assay and Transwell migration assay. The expression levels of molecular mechanisms related proteins MMP-2 and MMP-9 were assessed by Western blotting. Results The HepG2/mTOR^- cells with mTOR knockout expression were successfully constructed. COE significantly inhibited the proliferation of HepG2/mTOR^- cells in a concentration-dependent manner (P < 0.05). COE decreased the invasion and migration of HepG2/mTOR^- cells. The results of Transwell experiment indicated that COE (80 mg/L) significantly reduced the number of transmembrane cells (P < 0.05). And the expression levels of MMP2 and MMP9 protein were significantly reduced in the HepG2/mTOR^- cells after the treatment of COE. Conclusion COE can significantly inhibit the proliferation, invasion, and migration in the HepG2/mTOR^- cells. Our data reveal that COE is a potential chemotherapeutic drug in human hepatocellular carcinoma treatments via targeting mTOR signal pathway.

国家自然科学基金资助项目（81403232，81573656）；江苏省自然科学基金项目（BK20171290，BK2012686）；教育部博士点基金新教师类项目（20133250120003）