二甲双胍通过microRNA／mTOR通路抑制甲状腺未分化癌细胞增殖

Metformin inhibits the growth of anaplastic thyroid cancer cell by miRNA/mTOR pathway

目的探讨二甲双胍对阿霉素耐药的甲状腺未分化癌HTh74Rdox细胞生长的影响及其分子机制。方法二甲双胍作用于HTh74Rdox 48 h后，以光学显微镜观察细胞形态变化，四甲基偶氮唑盐（MTT）法检测细胞活力，以锚定蛋白Ⅴ-碘化丙啶双染的流式细胞术检测细胞凋亡情况，以实时荧光定量聚合酶链式反应（RT-PCR）检测抑癌microRNA的表达水平，以Western印迹检测一磷酸腺苷活化的蛋白激酶（AMPK）及哺乳动物雷帕霉素靶蛋白（mTOR）的表达水平；HTh74Rdox细胞转染miRNA inhibitors 24 h，继以二甲双胍干预48 h后，以流式细胞术检测细胞凋亡率，以Western印迹检测AMPK和mTOR的蛋白表达水平。结果二甲双胍可呈剂量依赖性地抑制HTh74Rdox的细胞活力、促进细胞凋亡，并上调miR-34a、miR-101、miR-125b及miR-138的表达，同时上调磷酸化（p-）AMPK、抑制p-mTOR的表达；以miR-inhibitors共转阻断miR-34a、miR-101、miR-125b、miR-138的表达后，可逆转二甲双胍对HTh74Rdox细胞的促凋亡效应以及对AMPK/mTOR通路蛋白的激活效应。结论二甲双胍通过上调阿霉素耐药的人甲状腺未分化癌HTh74Rdox细胞中抑癌基因miR-34a、miR-101、miR-125b、miR-138的表达，激活能量代谢信号通路AMPK/mTOR，发挥抑制肿瘤细胞生长、促进肿瘤细胞凋亡的作用。

ObjectiveTo investigate the effect of metformin on the growth of human anaplastic thyroid cancer cell HTh74Rdox which is doxorubicin resistant.MethodsThe HTh74Rdox was treated with different concentrations of metformin for 48 h. Cell morphology was observed by microscope, cell viability was tested by methylthiazoletetrazolium （MTT）, cell apoptosis by annexin Ⅴ and propidium iodide double staining, the anti-oncogenic miRNA was assayed by realtime fluorescence quantitative PCR （RT-PCR）, and the adenosine monophosphate-activated protein kinase （AMPK）/mammalian target of rapamycin （mTOR） signaling pathway tested by western blot. Furthermore, the anti-oncogenic miRNAs were knockdown by miRNA inhibitors （miR-34a, miR-101, miR-125b, and miR-138 inhibitors） and the cells were treated by metformin for 48 h, after that, cell apoptosis was detected by annexin Ⅴ and propidium iodide double staining, the expression of protein related to AMPK/mTOR signaling pathway was detected by western blot.ResultsMetformin inhibited the growth of human anaplastic thyroid cancer cell HTh74Rdox in a concentration-dependent manner, the cell apoptosis was induced by metformin, and there was a significantly lower expression of miR-34a, miR-101, miR-125b, and miR-138 in the HTh74Rdox. However, the four above miRNAs were upregulated by metformin, and AMPK/mTOR pathway was also activated by metformin. When these miRNAs were suppressed by miR-inhibitors （miR-34a, miR-101, miR-125b, miR-138 inhibitors）, the stimulating effect of apoptosis and AMPK/mTOR pathway by metformin were reversed.ConclusionMetformin significantly suppresses cell viability of human anaplastic thyroid cancer cell HTh74Rdox, and stimulates AMPK/mTOR pathway and apoptosis by upregulating the expressions of miR-34a, miR-101, miR-125b, miR-138 in HTh74Rdox cell.