The Roles And Mechanisms Of MiR-342-3p In Regulation Of Glycolysis In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the most common malignant tumor in liver which is prevalent all over the world.The latest epidemic statistics show that HCC is the fifth most common human cancer and the third lethal cause of cancer death in the world.HCC has a certain gender difference,standing for the third most common cancer in man and eighth in women.There are various features in HCC such as faster growth,higher malignance,vascular invasion,intrahepatic and extrahepatic metastasis,and recurrence.The prognosis of HCC is poor,and the 5-year survival rate is only 3%-25%,seriously threating the health of human beings.At present,the pathogenesis and mechanism of HCC are not fully elucidated.Therefore,it is very important and urgent to find new molecular targets and signaling pathways,which having crucial scientific and clinical significance.MicroRNAs(miRNAs)are a kind of single chain,approximately 14-24nucleotides in length,non-coding small RNA.By binding to the target genes3'-untranslation region(3'-UTR)specially,miRNAs lead to degradation or translation inhibition of target genes.They take part in many physiological and pathological processes such as cell cycle,proliferation,differentiation and apoptosis.A large number of studies have shown that miRNAs play important role in the development of tumor as cancer promoter or suppressor.The dysfunction of miRNAs affects tumor progression.Therefore,miRNAs may be a potential molecular biomarker for tumor diagnosis and treatment.Metabolism is the most basic characteristic of living organisms.Cells metabolism consume much energy,and glucose metabolism is the main way for cells to obtain energy.In cells,glucose is metabolized through oxidative phosphorylation and glycolysis.In normal condition,after cellular uptake through glucose transporters(GLUTs),glucose undergoes a series of biological processes which generates pyruvate.Pyruvate is converted into carbon dioxide,water and ATP through TCA in mitochondria.Under the condition of hypoxia environment,glucose is metabolised in the way of glycolysis.When generating pyruvate,it interacts with lactate dehydrogenase which produces lactate,protons,and a small amount of ATP in the end.Tumor cells exhibit high level of glycolysis despite the presence of sufficient oxygen.This phenomenon is called aerobic glycolysis,also known as Warburg effect.It is observed firstly by Warburg.The reprogrammed metabolism way bypasses mitochondrial TCA cycle which generates ATP with lower efficiency,but at a faster rate.The glucose carbon is transfered to synthesize nucleotides,lipids and proteins meeting the demands of tumor cells growth.Therefore,aerobic glycolysis may be a potential target for cancer therapy.Recently,miRNAs have been shown to play important roles in glucose metabolism in cancer proliferation,invasion,migration,and angiogenesis.In previous work,researchers identified the suppressive role of miR-342-3p in controlling the progression of liver cancer,lung cancer,cervical cancer,colonal cancer,and bladder cancer.In conclusion,we speculate that miR-342-3p may inhibit hepatocellular carcinoma progression by suppressing glycolysis.Cell biology and molecular biology methods including cell trasfection,RNA interference,qRT-PCR and luciferase reporter assay are used in cell experiment in vitro and animal experiment in vivo in this study.We aim to determine whether miR-342-3p regulates Warburg effect in HCC.And we also explore the potential mechanism.Part 1 Role of miR-342-3p regulating glycolysis in hepatocellular carcinoma.Objective:To investigate the correlation between glycolysis and miR-342-3p in regulating hepatoma cells proliferation.Methods:Cell transfection and glycolysis inhibitor 2-deoxidation-D-glucose(2-DG)were used to treat hepatoma cells.The proliferation curve determined by cell counting kit 8 and colony formation assay were performed to illustrate the cell growth ability.The expression level of miR-342-3p was analyzed by qRT-PCR.Results:1.The proliferation curve and colony formation assay showed that the proliferation ability of Hep G2 cells decreased after transfected with miR-342-3p compared with the control group.(P<0.01)2.The proliferation curve and colony formation assay showed that the proliferation ability of MHCC97H cells decreased after transfected with miR-342-3p compared with the control group.(P<0.01)3.The proliferation curve and colony formation assay showed that glycolytic inhibitor 2-DG impaired the ability of miR-342-3p mimics to inhibit proliferation in Hep G2 hepatoma cells.4.The proliferation curve and colony formation assay showed that glycolytic inhibitor 2-DG impaired the ability of miR-342-3p mimics to inhibit proliferation in MHCC97H hepatoma cells.Conclusions:1.miR-342-3p inhibits proliferation of human hepatoma cells.2.Aerobic glycolysis is critical for regulating hepatoma cell proliferation by miR-342-3p.Part 2 Effect of miR-342-3p on the target gene IGF-1RObjective:To explore the interaction between miR-342-3p and its target gene and signaling transduction downstream.Methods:Western blot and RT-PCR were used to determine the influence of miR-342-3p on the target gene and signaling pathway downstream after transfected with miR-342-3p control,mimics and inhibitor.The IGF-1R 3'-UTR wild type or IGF-1R 3'-UTR mutant plasmid DNA were cotransfected with miR-342-3p control or miR-342-3p mimics,then luciferase report assay was used to identify the hepatoma cell activity.The relationship between miR-342-3p and its target gene IGF-1R was confirmed in the end.Results:1.TargetScan and miRanda were performed to predict the target genes of miR-342-3p,and western blot was used to confirm the result.It reflected that IGF-1R was the target gene of miR-342-3p.miR-342-3p inhibited the expression of IGF-1R.2.miR-342-3p mimics inhibited the expression of IGF-1R and phosphorylated IGF-1R.Phosphorylated AKT and the expression of GLUT1were further inhibited in human heptoma cells Hep G2 and MHCC97H.3.miR-342-3p inhibitor promoted the expression of IGF-1R and phosphorylated IGF-1R.Phosphorylated AKT and the expression of GLUT1were further upregulated in human heptoma cells Hep G2 and MHCC97H.4.miR-342-3p mimics inhibited mRNA expression of IGF-1R in human heptoma cells Hep G2 and MHCC97H.(P<0.01)5.miR-342-3p inhibitor promoted mRNA expression of IGF-1R in human heptoma cells Hep G2 and MHCC97H.(P<0.01)6.Luciferase report assay showed that IGF-1R was the target gene of miR-342-3p.Compared with the control group,luciferase report gene activity decreased significantly when cotransfected with IGF-1R 3'UTR wild type plasmid and miR-342-3p mimics in human heptoma cells Hep G2 and MHCC97H.Compared with the control group,luciferase report gene activity had no difference when cotransfected with IGF-1R 3'UTR mutant plasmid and miR-342-3p mimics in human heptoma cells Hep G2 and MHCC97H.(P<0.01)Conclusions:1.IGF-1R is the target gene of miR-342-3p.2.miR-342-3p inhibits PI3K/AKT/GLUT1 signal pathway transduction mediated by IGF-1R.Part 3 Investigation of miR-342-3p regulating hepatocellular carcinoma growth through IGF-1R mediated glycolysis.Objective:To explore whether miR-342-3p regulating hepatocellular carcinoma growth through IGF-1R mediated glycolysis in HCC cells.Methods:Cell transfection and RNA interference were used to treat hepatoma cells.The proliferation curve determined by cell counting kit 8 and colony formation assay were performed to illustrate the cell growth ability.Glucose uptake,ATP concertration,lactate generation,extracellular acidification rate,and oxygen consumption rate were performed to show the cell glucose metabolism.Results:1.miR-342-3p inhibited the growth and colony formation of human hepatoma cells.IGF-1R knockdown impaired the ability of miR-342-3p mimics to inhibit proliferation in Hep G2 hepatoma cells.(P<0.05)2.miR-342-3p inhibited glucose uptake,ATP concertration and lactate generation of human hepatoma cells.IGF-1R knockdown impaired the ability of miR-342-3p mimics to inhibit glucose uptake,ATP concertration and lactate generation ability in Hep G2 hepatoma cells.(P<0.05)3.miR-342-3p inhibited the proliferation of Hep G2 cells,which could be reversed by IGF-1R reexpression in the miR-342-3p transfected cell lines.(P<0.05)4.miR-342-3p inhibited glucose uptake,lactate production and ATP generation.These effects were reversed by IGF-1R reexpression in the miR-342-3p transfected cells.(P<0.05)5.miR-342-3p inhibited extracellular acidification rate of human hepatoma cells.IGF-1R knockdown impaired the ability of miR-342-3p mimics to inhibit extracellular acidification rate.(P<0.05)6.miR-342-3p upregulated oxygen consumption rate of human hepatoma cells.IGF-1R knockdown impaired the ability of miR-342-3p mimics to upregulate oxygen consumption rate.(P<0.05)Conclusions:miR-342-3p suppresses hepatocellular carcinoma cells proliferation through inhibition of IGF-1R mediated glycolysis.Part 4 Role of miR-342-3p regulating glycolysis in vivo.Objective:To explore whether miR-342-3p regulating hepatocellular carcinoma growth through IGF-1R mediated glycolysis in vivo.Methods:For tumor assay in vivo,cells were injected subcutaneously into the hind flank of nude mice.An animal PET scanner was used for performing the glucose uptake ability.Lactate colorimetric assay kit was used to mesure lactate generation in tumor.Wstern blot was used to detect the expression level of related proteins in tumor.Results:1.The tumor-bearing nude mice were injected with ¹⁸F-FDG in order to perform PET scans.Compared with control group,the tumor group with stable expression of miR-342-3p exhibited lower ability of proliferation and glucose uptake.(P<0.01)2.Compared with the control group,the lactate production of tumor tissues decreased in the miR-342-3p overexpressed group.(P<0.01)3.Compared with the control group,the expression of pAKT,GLUT1,pIGF-1R,and IGF-1R was downregulated by miR-342-3p overexpression.(P<0.01)Conclusions:In vivo,PI3K/AKT/GLUT1 signaling pathway mediated by IGF-1R is involved in miR-342-3p inhibiting the proliferation and glycolysis in HCC.Conclusions:1.Aerobic glycolysis is critical for regulating hepatoma cell proliferation by miR-342-3p.2.miR-342-3p inhibits PI3K/AKT/GLUT1 signal pathway transduction mediated by IGF-1R.3.miR-342-3p suppresses hepatocellular carcinoma cells proliferation through inhibition of IGF-1R mediated glycolysis.4.In vivo,PI3K/AKT/GLUT1 signaling pathway mediated by IGF-1R is involved in miR-342-3p inhibiting the proliferation and glycolysis in HCC.