下调mTOR基因表达对人肝癌细胞SMMC-7721热疗增敏效应的影响

The effect of down-regulating the expression of mTOR on the thermosensitivity of human hepatoceUular carcinoma cell line SMMC-7721

目的观察下调哺乳动物雷帕霉素靶蛋白（mTOR）基因表达对人肝癌细胞SMMC-7721热疗敏感性的影响并初步探讨其作用机制。方法通过脂质体介导mTOR反义真核表达载体转染SMMC-7721细胞，分别采用RT—PCR和Western blot技术检测转染后roTOR基因mRNA及蛋白表达。SMMC-7721细胞经转染后给予热疗处理，采用CCK-8法检测细胞增殖活性，采用平板克隆实验检测克隆形成率，采用细胞划痕实验检测迁移能力，采用流式细胞仪检测细胞凋亡及周期变化情况。结果SMMC-7721细胞转染后其mTOR基因mRNA表达（0．23±0．01）及蛋白表达（0．31±0．02）均明显降低（P〈0．05），提示反义载体能有效下调SMMC-7721细胞mTOR基因表达。SMMC-7721细胞经转染后给予热疗处理，发现细胞增殖活性、克隆形成能力及细胞迁移能力均明显降低（P〈0．05）。通过流式细胞仪检测发现，实验组SMMC-7721细胞经热疗后其凋亡率[（52．27±3．72）％]较阴性对照组凋亡率[（28．93±2．51）％]明显增高，其细胞周期呈现S期比例增加（P〈0．05）及生长阻滞现象。结论下调mTOR基因表达能提高人肝癌细胞SMMC-7721的热疗敏感性，其作用机制可能与诱导细胞凋亡及S期生长阻滞有关。

Objective To investigate the effect of hyperthernfia on human hepatocellular carcinoma cell line SMMC-7721 after down-regulating the expression of roTOR, and its possible mechanisms. Methods An antisense roTOR （mammalian target of rapamycin） gene eukaryotic expression vector was transfected into SMMC-7721 cells. The expression of mTOR mRNA and protein were detected using RT-PCR and Western blotting, respectively. Hyper- thermia was applied after the transfection, and the vitality of cell proliferation was evaluated using CCK-8 assays and the clone formation rate was determined by colony-forming assays. The migration of SMMC-7721 cells was measured using scratch assays. Apoptosis and the cell cycle were analyzed by flow cytometry. Results The expression of roTOR mRNA and protein were significantly decreased after transfection, indicating that the antisense vector could down-regulate the mTOR gene effectively. The proliferation, clone formation and migration of SMMC-7721 cells all were decreased markedly by hyperthermia after transfection. Flow cytometry showed that the rate of apoptosis was significantly increased. The number of cells in the S phase was increased and the cell cycle was induced to arrest at the S phase. Conclusions Down-regulating the expression of roTOR can increase the thermosensitivity of SMMC-7721 cells. The mechanism involves increased apoptosis and S phase arrest.