Mechanism Of PDHA1 Affecting Cell Cycle Arrest And Apoptosis Of Hepatocellular Carcinoma Through ROS/PI3K/Akt Pathway

Hepatocellular carcinoma(HCC)accounts for 85-90%of primary liver cancer,and it is one of the most common cancers.The incidence rate ranks fifth among the world’s malignant tumors,and lacks effective treatment and prevention methods.With an extremely high recurrence rate and metastasis rate,the overall prognosis of patients is very poor and has become the third leading cause of cancer death worldwide.In China,the incidence and mortality of liver cancer are high,and the mortality rate ranks second in the cause of cancer death.About 383,000 people die of liver cancer each year,accounting for 51%of the world’s deaths due to liver cancer.Cancer is a multi-step,multi-gene participation process.Robert A.Weinberg,a pioneer in cancer research,summarized ten major biological features in the process of tumor development.Abnormal metabolism has become a major feature of tumor tissue that distinguishes it from normal tissues.With continuous research,its role in the development of tumors is increasingly valued.Abnormal tumor metabolism not only provides energy for the rapid proliferation of tumor cells,synthetic raw materials,and a large number of reducing substances,but also affects tumor signal pathway conduction and gene expression,and then participates in the regulation of tumor cell growth,apoptosis resistance and migration attacks.In recent years,the abnormal metabolism of glucose in tumor cells has been the most in-depth metabolic research.Glucose is first converted into pyruvate in the cytoplasm.When there is sufficient oxygen,pyruvate enters the mitochondria and participates in the oxidative phosphorylation of the tricarboxylic acid.The production of adenosine triphosphate(ATP)provides energy for the vital activity of the cell.One mole of glucose produces 36 moles of ATP through the mitochondria oxidizes phosphophosphate pathway.In the absence of oxygen,pyruvate is converted to lactic acid by lactate dehydrogenase and excreted extracellular.One mole of glucose produces two moles of ATP by glycolysis.A German biochemist Otto Warburg discovered in 1924 that even in the presence of oxygen,tumor cells preferentially use glycolysis as a means of energy supply,and most of the pyruvate transformed from glucose is catalyzed by lactate dehydrogenase.Since lactic acid is released to the outside,only a small portion of pyruvate enters the mitochondria and participates in oxidative phosphorylation.This metabolic pattern is called aerobic glycolysis,also known as the Warburg effect.With the deepening of research,aerobic glycolysis has played an important regulatory role in the occurrence,development,migration and invasion of tumors.In recent years,a number of studies have shown that targeting molecules inhibit glycolysis and enhance oxidative phosphorylation to achieve the purpose of inhibiting tumors,allowing researchers to gradually shift their focus from aerobic glycolysis to regulated oxidative phosphorylation and thus alter the behavior biology of tumor cells.Pyruvate dehydrogenase complex(PDHc)is present in mitochondria and catalyzes the conversion of pyruvate to acetyl-CoA to enter the tricarboxylic acid(TCA)cycle to participate in oxidative phosphorylation,which determines whether cells participate in aerobic oxidation or glycolysis and is the central control site of glucose metabolism.Several studies have shown that PDHc plays an important role in the rapid growth of tumor cells,apoptosis resistance,migration and invasion.The pyruvate dehydrogenase E1α subunit(PDHA1)is the major regulatory site of PDHc activity.Phosphorylation and dephosphorylation determine whether the activity of PDHc can be normally exerted,thereby regulating the mitochondria TCA cycle and metabolic flux of glycolysis.Related studies have shown that PDHA1 is abnormally expressed in a variety of tumor tissues,and changes the metabolic pattern of glucose in tumor cells by affecting the activity of PDHc and is closely related to tumor growth,sensitivity to radio-chemotherapy,drug resistance,and prognosis.Deletion of PDHA1 in prostate and ovarian cancer cells can inhibit oxidative phosphorylation,enhance glycolysis,and reduce ROS levels,which in turn lead to rapid proliferation of tumor cells.Conversely,there is no report that overexpression of PDHA1 gene affects the metabolism and growth of tumor cells has been reported at present.In view of this,this experiment takes PDHA1 as the starting point,aiming to investigate the effect of PDHA1 on tumor cell growth and its molecular mechanism from the perspective of glucose metabolism,and then to understand the relationship between changes in energy metabolism and tumor proliferation and apoptosis regulation.Part 1 The expression and prognosis of PDHA1 in hepatocellular carcinomaObjective:The expression level of PDHA1 was detected in hepatocellular carcinoma and matched paired tissues,and the relationship between the expression level of PDHA1 and the clinicopathological features of liver cancer was analyzed.Methods:1.Western blot and qRT-PCR methods were used to detect the expression of PDHA1 in 15 cases of HCC tissues and adjacent tissues.2.Immunohistochemistry was used to detect the protein expression of PDHA1 in 75 cases of hepatocellular carcinoma tissues and matched paracancerous tissues.The relationship between the expression of PDHA1 and clinicopathological features and prognosis of hepatocellular carcinoma was analyzed.Results:1.The expression level of PDHA1 mRNA and protein in 15 cases of HCC tissues was significantly lower than that in paracancerous tissues.2.Among the 75 cases of hepatocellular carcinoma and paracancerous tissues,the expression of PDHA1 in 52 cases of hepatocellular carcinoma was lower than that in paracancerous tissues.The level of PDHA1 expression was significantly correlated with histological grade,tumor size and TNM staging.The lower the PDHA1 protein expression,the worse the patient’s prognosis.Part 2 Effect of PDHA1 overexpression on glucose metabolism in hepatoma cellsObjective:1.Construction of PDHA1 overexpression lentiviral vector,the establishment of stable PDHA1 over-expression of liver cancer cell lines.2.To study the effect of overexpression of PDHA1 gene on glucose metabolism in hepatoma cell lines SMMC-7721 and HepG2.Methods:1.Western blot detection PDHA1 protein levels in a variety of liver cancer cell lines.2.Overexpression of lentiviral vector targeting PDHA1 gene was constructed,and the virus was packaged to infect hepatocellular carcinoma cells.Puromycin was selected to establish a stable upregulation of PDHA1 cell line.Western blot and qRT-PCR methods were used to verify the overexpression effect and enter the subsequent experiments.3.The effect of PDHA1 overexpression on the glycometabolism of hepatocellular carcinoma cells was analyzed by measuring cell PDH enzyme activity,citrate synthase activity,glucose consumption,ATP production and lactic acid yield.Results:1.PDHA1 is expressed in hepatoma cell lines SMMC-7721,MHCC97-H,BEL-7402,HepG2,QGY-7703 and QGY-7701.Based on the expression,we select HepG2 and SMMC-7721 whose underlying expression levels are low as targets for PDHA1 overexpression.The lentiviral vector transfected with PDHA1 was transfected.After a week of puromycin selection,a stable cell line was established.Validation of PDHA1 overexpression was confirmed by Western blot and qRT-PCR.2.After upregulation of PDHA1 expression in SMMC-7721 and HepG2 cells,the cellular PDH enzyme activity increased,glucose consumption decreased,citrate synthase activity increased,ATP production increased,lactic acid formation decreased,indicating that the level of glucose oxidative phosphorylation increased,glycolysis suppressed.Part 3 Overexpression of PDHA1 induces cell cycle arrest and apoptosis in hepatoma cells via ROS/P13K/Akt pathwayObjective:1.To study the effect of overexpression of PDHA1 gene on the biological behavior of hepatocellular carcinoma cells.2.To explore the molecular mechanism of upregulating PDHA1 induced cell cycle arrest and apoptosis through ROS/P13K/Akt signaling pathway.Methods:1.Through CCK8,clone formation experiments to understand the effect of PDHA1 upregulation on proliferation of hepatoma cells.2.Edu assay detects the effect of PDHA1 overexpression on DNA synthesis ability.3.Optical microscopy understands the morphological changes of the hepatoma cells after up-regulation of PDHA1 expression.4.Hochest 33452 and flow cytometry were used to detect the effect of overexpression of PDHA1 on the apoptosis of hepatocellular carcinoma cells.The changes of apoptosis related proteins were detected by Western blot.5.Flow cytometry was used to detect the effect of PDHA1 upregulation on the cell cycle distribution,and Western blot was used to detect the changes of the cycle related proteins.6.Fluorescence microscopy and fluorescence microplate readers were used to detect the effect of PDHA1 upregulation on ROS levels.7.Western blot was used to detect the effect of different ROS levels on the P13K/Akt pathway.8.Western blot detected changes in PI3K/Akt pathway after up-regulation of PDHA1 expression in combination with Akt agonist SC79.9.Flow cytometry detected the apoptosis of hepatoma cells after upregulation of PDHA1 expression and Akt agonist SC79,and the changes of apoptosis related proteins were detected by Western blot.10.The flow cytometry was used to detect the upregulation of PDHA1 expression in hepatocellular carcinoma cells and the change of cell cycle of Akt agonist SC79.Western blot was used to detect the changes of the cycle-related proteins.Results:1.Through CCK8 and Edu experiments,it was found that the ability of cells to synthesize DNA after PDHA1 overexpression in SMMC-7721 and HepG2 cells was reduced,and proliferation was inhibited.In colony formation experiments,it was observed that the colony forming ability of the cells was decreased after overexpression of the PDHA1 gene.2.The overexpression of PDHA1 in cells increased the ROS level and P13K/Akt activity was inhibited.After N-acetylcysteine decreased the ROS level,the inhibition of PI3K/Akt was reversed,confirming that ROS was upstream events of overexpression of PDHA1 led to PI3K/Akt inhibition.3.PDHA1 upregulation of cells combined with Akt agonist SC79 resulted in amelioration of apoptosis and cycle arrest.It was found that PI3K/Akt signaling pathway played an important regulatory role in upregulating PDHA1 induced cell cycle arrest and apoptosis.Part 4 Effect of PDHA1 overexpression on the tumorigenicity of orthotopic hepatocellular carcinoma in nude miceObjective:The effect of overexpression of PDHA1 on the tumorigenicity,glucose metabolism and apoptosis of hepatocellular carcinoma cell lines was observed by tumor formation in nude mice.Methods:1.Establish a xenograft tumor model in nude mice and observe the effect of overexpression of PDHA1 on tumor formation in hepatoma cells.2.Immunohistochemistry method was used to detect the expression levels of citric acid synthase and isocitrate dehydrogenase,which are the rate-limiting enzymes of the TCA in tumor tissue of nude mice.3.Tunel assay to detect tumor cell apoptosis in nude mice.Results:1.Tumor formation experiments in nude mice suggest that overexpression of PDHA1 can inhibit the tumorigenicity of SMMC-7721 cells in vivo.2.Immunohistochemical staining showed that the staining of citrate synthase and isocitrate dehydrogenase in transplanted tumors overexpressing PDHA1 was enhanced,indicating that TCA cycle metabolism was enhanced.3.Tunel results showed that the overexpression of PDHA1 group increased the apoptosis of tumor tissue in nude mice.3.Tunel results showed that the overexpression of PDHA1 group increased the apoptosis of tumor tissue in nude mice.Conclutions1.PDHA1 is lowly expressed in HCC tissues.The expression level of PDHA1 is related to tumor size,pathological grade,TNM stage and prognosis.The lower the expression level,the worse the prognosis.2.Overexpression of PDHA1 in hepatocarcinoma cells enhanced PDH enzyme activity,increased mitochondrial oxidative phosphorylation rate,and inhibited glycolysis.3.In vitro experiments confirmed that overexpression of PDHA1 in hepatoma cells induced cell cycle arrest and apoptosis through ROS/P13K/Akt pathway.4.In vivo nude mice tumorigenesis confirmed that overexpression of PDHA1 can inhibit tumorigenicity of hepatocellular carcinoma cells,enhance TCA cycle metabolism,and promote apoptosis.