二甲双胍通过REDD1-mTOR信号通路抑制肝癌细胞增殖的机制研究

摘要/Abstract

摘要： 目的探讨二甲双胍(MET)通过发育及DNA损伤反应调节基因1(REDD1)-雷帕霉素靶蛋白(mTOR)信号通路抑制肝癌细胞增殖的机制。方法 MTT法检测不同浓度MET(0.0、2.5、5.0、10.0 mmol/L)对HepG2细胞增殖的影响。选择10.0 mmol/L的MET进行后续实验,将其分为Control组、MET组、sh-NC组、sh-REDD1组;qRT-PCR法检测各组HepG2细胞中REDD1、mTOR、p70s6k、Cyclin D1 mRNA的表达;平板克隆实验检测各组HepG2细胞增殖;流式细胞仪检测各组HepG2的细胞周期和凋亡;WB检测各组HepG2细胞中REDD1、mTOR、p70s6k、Cyclin D1蛋白质的表达。结果 MET组G1期细胞比例(81.25%±10.35%)、凋亡率(42.55%±4.52%)、REDD1 mRNA(7.52±0.80)和蛋白质表达(0.86±0.13)高于Control组(36.58%±8.49%、1.87%±0.64%、1.00±0.33、0.27±0.08),S期(11.19%±2.58%)、G2期细胞比例(7.56%±2.12%)、克隆数[(53.41±9.28)个]、mTOR mRNA(0.36±0.08)和蛋白质表达(0.52±0.15)、p70s6kmRNA(0.47±0.14)和蛋白质表达(0.43±0.09)、Cyclin D1mRNA(0.53±0.13)和蛋白质表达(0.48±0.11)低于Control组[30.87%±4.28%、32.55%±4.36%、(118.43±12.92)个、0.99±0.12、1.17±0.17、1.00±0.21、0.94±0.14、1.01±0.17、1.05±0.17,P＜0.05];与MET组、sh-NC组相比,sh-REDD1组G1期细胞比例、凋亡率、REDD1 mRNA和蛋白质表达降低,S期、G2期细胞比例、克隆数、mTOR mRNA和蛋白质表达、p70s6k mRNA和蛋白质表达、Cyclin D1 mRNA和蛋白质表达升高(P＜0.05)。结论 MET可抑制HepG2细胞的增殖活性,其可能的分子机制是通过REDD1-mTOR信号通路发挥抑制肝癌细胞增殖作用。

关键词: 二甲双胍, 肝癌, 发育及DNA损伤反应调节基因1, 雷帕霉素靶蛋白, 细胞增殖

Abstract: Objective To investigate the mechanism by which metformin (MET) inhibits the proliferation of liver cancer cells by regulating the regulated in development and DNA damage response 1 (REDD1) - mammalian target of rapamycin (mTOR) signaling pathway. Methods MTT assay was used to detect the effects of different concentrations of MET (0.0, 2.5, 5.0, 10.0 mmol/L) on the proliferation of HepG2 cells. 10.0 mmol/L MET was selected for subsequent experiments and the cells were separated into Control group, MET group, sh-NC group, and sh-REDD1 group. QRT-PCR method was applied to detect the expression of REDD1 mTOR p70s6k and Cyclin D1 mRNA in HepG2 cells in each group. The plate cloning experiment was applied to detect the proliferation of HepG2 cells in each group. Flow cytometry was applied to detect the cell cycle of HepG2 in each group. WB was applied to detect the expression of REDD1, mTOR, p70s6k, and Cyclin D1 proteins in HepG2 cells of each group. Results The proportion of G1 phase cells (81.25%±10.35%), apoptosis rate(42.55%±4.52%), REDD1 mRNA (7.52±0.80) and protein expression (0.86±0.13) in the MET group were higher than those in the Control group 36.58%±8.49%,1.87%±0.64%,1.00±0.33,0.27±0.08, the proportions of S phase(11.19%±2.58%) and G2 phase cells(7.56%±2.12%), number of clones(53.41±9.28 number), mTOR mRNA (0.36±0.08) and protein (0.52±0.15), p70s6k mRNA (0.47±0.14) and protein (0.43±0.09) , and Cyclin D1 mRNA (0.53±0.13) and protein expression (0.48±0.11) were lower than those in the control group (30.87%±4.28%, 32.55%±4.36%, 118.43±12.92 number, 0.99±0.12, 1.17±0.17, 1.00±0.21, 0.94±0.14, 1.01±0.17, 1.05±0.17) (P<0.05). Compared with the MET group and sh-NC group, proportion of G1 phase cells, apoptosis rate, REDD1 mRNA and protein expression in the sh-REDD1 group were lower, the proportions of S phase and G2 phase cells, number of clones, mTOR mRNA and protein, p70s6k mRNA and protein, and Cyclin D1 mRNA and protein expression were higher (P<0.05). Conclusion MET can inhibit the proliferation activity of HepG2 cells, and its possible molecular mechanism is to exert an inhibitory effect on liver cancer cell proliferation through the REDD1-mTOR signaling pathway.

Key words: Metformin, Liver cancer, Regulated in development and DNA damage response 1, Mammalian target of rapamycin, Cell proliferation

王燕, 李琴, 李军军, 张小丽, 谭丽玲. 二甲双胍通过REDD1-mTOR信号通路抑制肝癌细胞增殖的机制研究[J]. 肝脏, 2025, 30(12): 1668-1673.

WANG Yan, LI Qin, LI Jun-jun, ZHANG Xiao-li, Tan Li-ling. The mechanism of metformin inhibiting liver cancer cell proliferation through the REDD1-mTOR signaling pathway[J]. Chinese Hepatolgy, 2025, 30(12): 1668-1673.

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