摘要目的:探讨轻甲硫氨酸限制(10%MR)对肝癌细胞增殖能力的影响及其机制。方法:采用对照短发夹RNA(shCtrl)及脂肪酸合酶(FASN)短发夹RNA (shFASN)慢病毒感染HuH7和HepG2，构建对照组和shFASN组细胞；采用细胞计数试剂盒(CCK-8)、克隆形成分析细胞增殖能力；采用流式细胞仪分析细胞周期变化；采用蛋白质免疫印迹分析增殖细胞核抗原(PCNA)、FASN、哺乳动物雷帕霉素靶蛋白(mTOR)、蛋白激酶B(Akt)和磷酸化蛋白激酶B(pAkt)的蛋白水平；分别采用荧光定量聚合酶链式反应(PCR)、亚硫酸氢盐测序、油红染色和Bodipy染色分析10%MR对FASN的mRNA、FASN的CpG岛甲基化及脂质累积的影响。组间比较采用 t检验。 结果:10%MR处理组HuH7细胞活性高于对照组(0.666±0.013比0.510±0.007， t＝－26.50， P<0.001)；处在G 2期的细胞比例高于对照组[(19.8±1.3)%比(12.1±1.8)%， t＝－5.91， P<0.01]；PCNA水平高于对照组(1.280±0.051比0.447±0.067， t＝－17.07， P<0.001)；mTOR蛋白表达高于对照组(1.161±0.277比0.478±0.061， t＝－4.17， P<0.05)；pAkt水平高于对照组(0.938±0.194比0.357±0.169， t＝－3.90， P<0.05)；FASN的CpG岛甲基化水平低于对照组(0.233±0.252比0.833±0.153， t＝3.53， P<0.05)；FASN的mRNA高于对照组(1.360±0.137比1.003±0.081， t＝－4.49， P<0.01)；FASN的蛋白高于对照组(0.915±0.118比0.648±0.034， t＝－3.76， P<0.05)。ShFASN组HuH7细胞活性低于对照组(0.303±0.019比0.414±0.018， t＝10.25， P<0.001)；G 2期的细胞比例低于对照组[(3.5±1.4)%比(7.6±1.4)%， t＝3.69， P<0.05]；mTOR蛋白水平低于对照组(0.276±0.079比0.646±0.068， t＝6.17， P<0.01)；pAkt水平低于对照组(0.296±0.016比0.494±0.051， t＝6.37， P<0.01)。10%MR处理的shFASN组HuH7细胞活性低于10%MR处理的shCtrl组(0.369±0.003比0.495±0.007， t＝41.06， P<0.001)；G 2期的细胞比例低于10%MR处理的shCtrl组[(11.1±0.4)%比(19.4±2.8)%， t＝5.05， P<0.01]；mTOR蛋白水平低于10%MR处理的shCtrl组(0.419±0.187比0.939±0.072， t＝4.49， P<0.05)；pAkt水平低于10%MR处理的shCtrl组(0.421±0.060比0.623±0.060， t＝4.11， P<0.05)。 结论:轻甲硫氨酸限制通过上调FASN的表达，加速细胞周期，进而促进肝癌细胞增殖。

abstractsObjective:To explore the effects of mild methionine restriction (10% MR) on proliferation of hepatoma cells and mechanism.Methods:To construct the control and stable fatty acid synthase (FASN) knockdown hepatoma cells, HuH7 and HepG2 were infected with control short hairpin RNA (shCtrl) and shFASN. The cell counting kit (CCK-8) and clonal formation assays were used to detect proliferation of hepatoma cells. The changes of cell cycle were analyzed by flow cytometry. The expression of proliferating cell nuclear antigen (PCNA), FASN, mammalian target of rapamycin (mTOR), protein kinase B (Akt), and phosphorylated protein kinase B (pAkt) was analyzed by Western blotting. The effects of 10%MR on FASN mRNA, methylation of CpG island in FASN, and lipid accumulation were analyzed by fluorescence quantitative polymerase chain reaction (PCR), bisulfite sequencing, and oil red staining and Bodipy staining, respectively.Results:As compared with control group, 10% MR-treated HuH7 cells had higher cell viability (0.666±0.013 vs. 0.510±0.007, t=-26.50, P<0.001), higher proportion of G 2 phase cells [(19.8±1.3)% vs. (12.1±1.8)%, t=-5.91, P<0.01], higher PCNA protein (1.280±0.051 vs. 0.447±0.067, t=-17.07, P<0.001), higher mTOR protein (1.161±0.277 vs. 0.478±0.061, t=-4.17, P<0.05), higher pAkt level (0.938±0.194 vs. 0.357±0.169, t=-3.90, P<0.05), lower methylation of CpG island in FASN (0.233±0.252 vs. 0.833±0.153, t=3.53, P<0.05), higher level of FASN mRNA (1.360±0.137 vs. 1.003±0.081, t=-4.49, P<0.01), higher level of FASN protein (0.915±0.118 vs. 0.648±0.034, t=-3.76, P<0.05). Compared with shCtrl group, shFASN group had lower cell viability (0.303±0.019 vs. 0.414±0.018, t=10.25, P<0.001), lower proportion of G 2 phase cells [(3.5±1.4)% vs. (7.6±1.4)%, t=3.69, P<0.05], lower mTOR protein (0.276±0.079 vs. 0.646±0.068, t=6.17, P<0.01), lower level of pAkt (0.296±0.016 vs. 0.494±0.051, t=6.37, P<0.01). Compared with shCtrl group treated with 10% MR, shFASN group treated with 10% MR had lower cell viability (0.369±0.003 vs. 0.495±0.007, t=41.06, P<0.001), lower proportion of G 2 phase cells [(11.1±0.4)% vs. (19.4±2.8)%, t=5.05, P<0.01], lower mTOR protein (0.419±0.187 vs. 0.939±0.072, t=4.49, P<0.05), lower level of pAkt (0.421±0.060 vs. 0.623±0.060, t=4.11, P<0.05). Conclusion:Mild methionine restriction accelerates cell cycle and promotes proliferation of hepatoma cells by upregulating the expression of FASN.