miR-130a-3p通过调控PDK1影响肝癌细胞糖代谢的研究

Effect of miR-130a-3p on Glucose Metabolism of Liver Cancer Cells by Regulating PDK1

目的探讨miR-130a-3p通过靶向糖代谢关键酶PDK1对肝癌细胞糖代谢以及增殖迁移的影响,为肝癌的诊断及治疗提供新的方向。方法qRT-PCR技术检测肝癌的临床组织和细胞中miR-130a-3p的表达,克隆形成、CCK-8、Transwell和细胞划痕实验检测细胞的增殖和迁移;Western印迹检测相关蛋白的表达;乳酸检测试剂盒,ATP检测试剂盒和PDH活性检测试剂盒分别检测细胞中乳酸的生成、ATP的生成和PDH的活性;双荧光素酶报告基因实验验证miR-130a-3p与PDK13′UTR靶向结合。结果在肝癌组织和细胞中miR-130a-3p呈低表达,并与患者肿瘤组织的大小和TNM分期有关;过表达miR-130a-3p能够增强肝癌细胞中PDH活性,抑制乳酸和ATP的生成,从而抑制肝癌细胞的增殖和迁移;miR-130a-3p可以靶向调控PDK1;抑制PDK1的表达,能够逆转miR-130a-3p过表达对细胞增殖和迁移能力的增强,此外对PDH活性、乳酸和ATP的影响也被逆转。结论miR-130a-3p通过靶向调控PDK1影响肝癌细胞糖酵解及恶性进展。

Objective To investigate the effect of miR-130a-3p on glucose metabolism,proliferation and migration of liver cancer cells by targeting PDK1,a key enzyme of glucose metabolism,so as to provide a new target for the diagnosis and treatment of liver cancer.Methods The expression of miR-130a-3p in clinical tissue samples and cell lines of hepatocellular carcinoma was detected by qRT-PCR.Cell proliferation was measured by clone formation and CCK-8.Cell migration was measured by transwell and wound healing assay.Western blotting was used to detect the expression of related proteins.The production of lactic acid and ATP in cells was assayed by lactate assay kit and ATP assay kit respectively,and the PDH activity in cells was assayed by PDH activity assay kit.The dual-luciferase reporter gene assay was used to verified the binding of miR-130a-3p to PDK13′UTR.Results The expression level of miR-130a-3p was lower in liver cancer tissues and cells,and it was related to the size of tumor tissues and TNM stage.Overexpression of miR-130a-3p could enhance the activity of PDH in hepatoma cells,reduce the production of lactic acid and ATP,and inhibit the proliferation and migration of hepatoma cells.miR-130a-3p could bind to PDK1 and regulate its expression.Inhibition of PDK1 expression could reverse the enhancing effects of miR-130a-3p overexpression on cell proliferation and migration,and the effects on PDH activity,production of lactic acid and ATP were also reversed.Conclusion miR-130a-3p affects glycolysis and malignant progression of hepatoma cells by targeting PDK1.