MiR-429 Regulates Aerobic Glycolysis Of Hepatocellular Carcinoma Through PI3K/AKT Pathway

Background:Hepatocellular carcinoma(HCC)is the most common malignant tumor of the liver.About 24% of newly diagnosed cancer cases and 30% of cancer related deaths in the world occur in China.Liver cancer is the fourth most common malignant tumor in China,with the second highest mortality rate.HCC grows fast,has a high malignancy,a low 5-year survival rate,it seriously endangers human health.HCC is a disease that causes the malignant proliferation of hepatocellular carcinoma cells due to the occurrence and development of multifactorial and multi-stage molecular events.Clarifying the key specific molecular mechanism of tumor pathogenesis is of great value for enriching the theoretical research of hepatocellular carcinoma and fundamentally improving the screening,diagnosis,prognosis evaluation and guiding treatment of tumor risk.Rapid proliferation of malignant tumors requires sufficient material and energy supply,and accompanied by significant changes in the metabolic pattern of tumor cells to adapt to tumor development,namely metabolic reprogramming.The molecular mechanism of initiating and regulating tumor metabolism reprogramming is very complex and has not yet been fully elucidated.Therefore,it is of great scientific significance to explore new molecular targets and signal pathways,as well as the role of aerobic glycolysis in liver cancer.Micro RNAs(miRNAs)are a class of endogenous single-stranded non-coding small RNA fragments with 18-25 nucleotides in length.Through specific binding with the 3 ’-UTR region of the target gene,they cause the degradation of the target gene or inhibit the translation of the target gene,thus regulating the basic life processes of cell proliferation,invasion,differentiation,apoptosis and so on.Mi R-429 is abnormally expressed in a variety of tumors and regulates different tumor processes as an oncogene or tumor suppressor gene.In our group’s previous study,miR-429 was found to act as an inhibitory factor in liver cancer,inhibiting the biological processes of liver cancer migration,invasion,EMT metastasis and so on.However,the role of miR-429 in aerobic glycolysis has not been reported.Therefore,this study aims to explore how miR-429 affects the acidic microenvironment of liver cancer cells by regulating the glucose metabolism of liver cancer cells,resulting in the loss or supply of tumor nutrition,and its function in aerobic glycolysis of liver cancer cells,and clarify its molecular mechanism.Objective:1.To clarify the effect of miR-429 on glycolysis and glycogen metabolism of hepatoma cells.2.Explore the molecular mechanism of miR-429 regulating glucose metabolism in hepatoma cells.Methods:1.The overexpression/knockdown level of miR-429 was detected by q RT-PCR.2.Glucose oxidase-peroxidase(GOD-POD)method was used to detect the effect of miR-429 expression on glucose uptake of hepatoma cells.3.The change of lactic acid production in HCC cell line caused by overexpression/knockdown of miR-429 was detected by lactate oxidase method.4.The influence of the change of miR-429 level on the acidity of liver cancer cell culture medium was detected with a p H meter.5.Western blotting method was used to detect the expression changes of miR-429overexpression/knockdown on key molecules of glycolysis pathway(glucose transporter 1,hexokinase 2,phosphofructose kinase 1,and pyruvate kinase 2)in hepatocellular carcinoma cell lines.6.DCFH-DA method was used to detect the regulation of the level of miR-429 on the reactive oxygen species in hepatoma cells.7.The effect of miR-429 expression on the activity of glycogen synthase(GCS)in hepatocellular carcinoma cells was detected by reducing coenzyme I(NADH)rate method.8.The effect of the change of miR-429 expression level on the activity of glycogen phosphorylase a(GPa)in hepatoma cells was detected by the reduction coenzyme II(NADPH)rate method.9.PAS staining and anthrone method were used to detect the effect of the change of miR-429 expression level on the glycogen in hepatoma cells.10.WB method was used to detect the effect of changes in miR-429 expression level on glycolysis,glycogen synthesis and PI3K/AKT pathway related molecular protein expression level.Results:1.The results of q RT-PCR showed that miR-429 was up-regulated 61967 times(P=0.0018)in HepG2-miR-429-mimic group cells and 67497 times(P=0.0058)in HCCLM3-miR-429-mimic group cells compared with NC-mimic group cells.Compared with NC-antagamir group cells,miR-429 down-regulated 59.7%(P=0.0024)and 50.3%(P=0.0007)in HepG2-antagamiR-429 group cells and HCCLM3-antagamiR-429 group cells respectively.2.The up-regulation of miR-429 reduces the glucose intake of HepG2 and HCCLM3 cells.And miR-429 down-regulated enhanced the glucose intake of HepG2 and HCCLM3 cells.3.After up-regulation of miR-429,the production of lactic acid in HepG2 and HCCLM3 cells decreased.Mi R-429 down-regulated and enhanced the production of lactic acid in HepG2 and HCCLM3 cells.4.HepG2 and HCCLM3 cells over-expressed and knocked down miR-429,and the p H value in the culture medium increased and decreased respectively.5.Overexpression of miR-429 in HepG2 and HCCLM3 cells,decreased expression of GLUT1,HKII,PFK1 and PKM2;The knockdown of miR-429 increased the expression of GLUT1,HKII,PFK1 and PKM2.6.Overexpression of miR-429 increased the intracellular ROS level of HepG2 and HCCLM3 cells,and knockdown of miR-429 decreased the intracellular ROS level.7.Overexpression of miR-429 increased the GCS activity of HepG2 and HCCLM3 cells,and knockdown of miR-429 decreased the GCS activity.8.miR-429 overexpression,GPa activity of HepG2 and HCCLM3 cells increased,miR-429 knockdown,GPa activity decreased.9.miR-429 overexpression of HepG2 and HCCLM3 cells increased PAS positive reaction and glycogen content;Mi R-429 knocks down the PAS positive reaction and glycogen content in HepG2 and HCCLM3 cells.10.Overexpression of miR-429 causes GSK3β in HepG2 and HCCLM3 cells protein level reduced,the protein level of p-GSK3β increases,and its knockdown leads to GSK3β protein level increased,the protein level of p-GSK3β decreased.The expression level of miR-429 in hepatoma cells was negatively correlated with the expression of CRKL.Overexpression of miR-429 significantly decreased the levels of PI3K85,PI3K110,p-AKT protein in liver cancer cells,and the AKT protein level remained unchanged.Down-regulation of miR-429 significantly increased the levels of PI3K85,PI3K110,p-AKT protein,and the AKT protein level remained unchanged.Conclusion:Through this research,we found that miR-429 can affect glucose uptake,lactic acid production,medium acidity and ROS production by regulating glycolysis pathway.The specific molecular mechanism is that miR-429 regulates PI3K/AKT signal pathway by targeting CRKL.We also found that miR-429 upregulates p-GSK3β、lower GSK3β expression can promote the production of glycogen by promoting the activity of glycogen synthase.The miR-429 knockdown is achieved by down-regulating p-GSK3β、increase GSK3β,it can inhibit the production of glycogen by reducing the activity of glycogen synthetase.Our research deepens the understanding of the molecular mechanism of miR-429 in the regulation of tumor metabolism reprogramming,clarifies the new relationship between miR-429 and glycolysis and glycogen synthesis,and provides a new idea for the clinical targeted treatment of liver cancer.