摘要
目的探讨磷脂酰肌醇-3-激酶/蛋白激酶B(PI3K/Akt)通过调节含己糖激酶结构域的蛋白1(HKDC1)对人肝癌HepG2细胞糖酵解、增殖和迁移的影响及机制。方法将体外培养的对数期人肝癌HepG2细胞,分为对照组、LY294002组和MK-2206组,RT-qPCR法检测各组细胞HKDC1 mRNA的表达水平,western blotting法分析各组细胞HKDC1蛋白的表达水平。将细胞分为si-NC组(转染si-NC)、si-HKDC1组(转染si-HKDC1),western blotting法分析各组细胞HKDC1蛋白的表达水平,CCK-8、5-乙炔基-2′-脱氧尿苷(EdU)实验检测各组细胞增殖活力和划痕,Transwell实验检测各组细胞迁移率,细胞外酸化率(ECAR)实验检测分析各组细胞的糖酵解和糖酵解能力,葡萄糖及乳酸含量测定实验检测各组细胞内葡萄糖及乳酸含量。将细胞分为对照组、LY294002组、过表达HKDC1+LY294002组、MK-2206组、过表达HKDC1+MK-2206组,葡萄糖及乳酸含量测定实验检测各组细胞内葡萄糖及乳酸含量。结果与对照组比较,LY294002组细胞HKDC1 mRNA表达明显降低(P<0.001),HKDC1蛋白表达降低(P<0.01);MK-2206组细胞HKDC1 mRNA表达降低(P<0.01),HKDC1蛋白表达明显降低(P<0.001)。与si-NC组相比,si-HKDC1组细胞HKDC1蛋白表达降低(P<0.001);与si-NC组相比,si-HKDC1组细胞增殖活力降低(P<0.05),EdU阳性细胞数明显减少(P<0.01);与si-NC组相比,si-HKDC1组细胞划痕愈合率明显降低(P<0.001),迁移细胞数明显减少(P<0.001);与si-NC组相比,si-HKDC1组细胞糖酵解和糖酵解能力明显减弱(P<0.01);与si-NC组相比,si-HKDC1组细胞内葡萄糖和乳酸含量明显降低(P<0.05)。与LY294002组相比,过表达HKDC1+LY294002组细胞内葡萄糖和乳酸含量明显升高(P<0.01);与MK-2206组相比,过表达HKDC1+MK-2206组细胞内葡萄糖和乳酸含量明显升高(P<0.001,P<0.01)。结论PI3K/Akt信号通路通过HKDC1促进人肝癌HepG2细胞糖酵解、增殖和迁移。
Objective To investigate the effects and mechanisms of PI3K/Akt on glycolysis,proliferation and migration of human hepatocellular carcinoma HepG2 cells by regulating HKDC1.Methods Log-phase human hepatocellular carcinoma HepG2 cells cultured in vitro were divided into negative control group,LY294002 group and MK-2206 group,and the expression level of HKDC1 mRNA of cells in each group was detected by RT-qPCR,and the expression level of HKDC1 protein of cells in each group was analyzed by western blotting.The cells were divided into si-NC group(transfected with si-NC)and si-HKDC1 group(transfected with si-HKDC1),and the expression level of HKDC1 protein in each group was analyzed by western blotting,the proliferation ability of cells in each group was detected by CCK-8 and EdU assays,the migration ability of cells in each group was detected by Wound healling and Transwell assays,the extracellular acidification rate(ECAR)assay experiment to analyze the glycolysis and glycolytic capacity of cells in each group,and glucose and lactate content assay experiment to detect the intracellular glucose and lactate content in each group.The cells were divided into control group,LY294002 group,overexpression of HKDC1+LY294002 group,MK-2206 group,and overexpression of HKDC1+MK-2206 group,and intracellular glucose and lactate contents were measured by glucose and lactate content assay.Results Compared with the control group,cells in the LY294002 group showed significantly lower HKDC1 mRNA expression(P<0.001)and lower HKDC1 protein expression(P<0.01);cells in the MK-2206 group showed lower HKDC1 mRNA expression(P<0.01)and significantly lower HKDC1 protein expression(P<0.001).Compared with the si-NC group,cellular HKDC1 protein expression was reduced in the si-HKDC1 group(P<0.001);compared with the si-NC group,cellular proliferation viability was reduced in the si-HKDC1 group(P<0.05),and the number of EdU-positive cells was significantly reduced(P<0.01);compared with the si-NC group,cellular wound healing rate was significantly reduced in the si-HKDC1 group(P<0.001),and the number of migrating cells was significantly reduced(P<0.001);cellular glycolysis and glycolytic capacity were significantly weakened in the si-HKDC1 group compared with the si-NC group(P<0.01);and intracellular glucose and lactic acid content was significantly reduced in the si-HKDC1 group compared with the si-NC group(P<0.05).The intracellular glucose and lactate contents were significantly higher in the overexpression HKDC1+LY294002 group compared with the LY294002 group(P<0.01);and the intracellular glucose and lactate contents were significantly higher in the overexpression HKDC1+MK-2206 group compared with the MK-2206 group(P<0.001,P<0.01).Conclusion The PI3K/Akt signaling pathway promotes glycolysis,proliferation and migration of human hepatocellular carcinoma HepG2 cells via HKDC1.
作者
刘峰
邓萍
闫光志
金刚
LIU Feng;DENG Ping;YAN Guangzhi;JIN Gang(The First Department Abdominal Surgical Oncology;Depurtment of Interventional Medicine,Jilin Cancer Hospitals Chang-chun 130012,China)
出处
《中国实验诊断学》
2024年第9期1079-1086,共8页
Chinese Journal of Laboratory Diagnosis
基金
吉林省科技厅自然科学基金项目(20200201610JC)。
