The Mechanism Of Aerobic Glycolysis And Glycogen Metabolism Regulated By ETV6 In HCC

Background:Hepatocellular carcinoma(Hepatocellular carcinoma,HCC)is the most common primary liver cancer with a poor prognosis.Because HCC is usually found in the clinical late,but the treatment of advanced HCC is very limited,in the past few decades,HCC mortality increased year by year,if liver cancer can early diagnosis and treatment,the survival rate of patients will greatly improve,therefore,effective early diagnosis and the development of new treatment demand.ETV6(ETS Variant Transcription Factor 6): also known as TEL oncogene,which encodes the ETS family transcription factor,ETS protein is a transcription factor,which can participate in the gene expression of cell proliferation,differentiation,apoptosis and other biological processes.The product of ETV 6 gene contains two functional domains: one for the PNT domain at the N end,involved in the interaction between protein and protein,and a DNA binding domain at the C end,ETV6 as a transcriptional repressor can bind with DNA sequence 5 ′-CCGGAAGT-3′,play a role in hematopoietic and malignant transformation,but the function and molecular mechanism has not been defined.Metabolic reprogramming: In the process of tumor occurrence and development,the infinite proliferation of tumor cells leads to them in the "starvation environment".In order to meet the needs of tumor cells for substances,energy and biological macromolecules,tumor cells reprogram by changing their metabolic pathway,which is called metabolic reprogramming.Even under the conditions of sufficient oxygen,after glucose is transported into cells through glucose transporter,it generates pyruvate through the glycolytic pathway and further generates lactate.The accumulated lactate is secreted outside the cell by monocarboxylate transporters to change the tumor microenvironment.This effect is called Warburg effect.The Warburg effect is one of the most widely studied patterns of cellular metabolic reprogramming.Glycogen is an energy storage substance that is easy to be mobilized in the process of energy metabolism.When glucose intake is insufficient,glycogen needs to be decomposed to provide energy for the body,including the process of glycogen synthesis and decomposition.Its metabolic pathway is also one of the common metabolic pathways of tumor cells.Objectives:1.Determine the effect of ETV6 on aerobic glycolysis and glycogen metabolism in HCC cells.2.Clarify the mechanism of ETV6 regulating aerobic glycolysis and glycogen metabolism in liver cancer cells.Methods:1.ETV6 was overexpressed and knocked down in HepG2 and HCCLM3 cells by transient transfection,and ETV6 expression was detected by Western blotting(WB)assay;2.The effect of glucose oxidase-peroxidase(Glucose oxidase-peroxidase,GOD-POD)on glucose uptake in HepG2 and HCCLM3 cells;3.The change of lactate secretion of HepG2 and HCCLM3 by lactate oxidase and quantification;4.Effect of ETV6 levels on the acidity of HepG2 and HCCLM3 cells;5.The effect of ETV6 level change on the generation of ROS by HepG2 and HCCLM3 was detected by chemical fluorescence method;6.The effect of ETV6 levels on the synthesis of HepG2 and HCCLM3 glycogen was detected by periodine-Schv reaction(periodic acid Schiff reaction,PAS)staining;7.The effect of ETV6 on glycogen content of HepG2 and HCCLM3 was determined by anthrone method;8.The effect of ETV6 expression was detected by reduced coenzyme I(n icotinamide adenine dinucleotide,NADH)on the activity of HepG2 and HCCLM3 glycoen synthase(glycogen synthase,GCS);9.The effect of ETV6 expression level on the activity of HepG2 and HCCLM3 glycogen phosphorylase(glycogen phosphorylase a,GPa)was detected;10.The effect of ETV6 changes on the expression of key molecules in glycolysis and glycogen metabolism was detected by WB;11.The effect of ETV6 changes on the expression of key molecules of the PI3 K / AKT pathway in HepG2 and HCCLM3 cells were determined by WB.Results:1.Overexpressing ETV6 in HepG2 and HCCLM3,ETV6 levels increased by 41.0%(P=0.0071)and 73.3%(P=0.0430),respectively;ETV6 knockdown in HepG2 and HCCLM3 decreased by 45.7%(P =0.0025)and 56.8%(P =0.0325);2.ETV6 overexpression increased glucose uptake in HepG 2 and HCCLM3 cells by 53.7%(P=0.0001)and 21.9%(P =0.0050),respectively;ETV6 knockdown decreased glucose uptake in HepG2 and HCCLM3 by 37.8%(P=0.0022)and 36.4%(P =0.0022),respectively;3.Compared with the control group,ETV6 overexpression increased HepG2 and HCCLM3 cells by 31.5%(P=0.0018)and 9.1%(P=0.0012),respectively,and ETV6 knockdown reduced HepG2 and HCCLM3 by 11.8%(P =0.0119)and 17.6%(P=0.0002);4.Compared with control group,ETV6 overexpression decreased the p H and acidity of HepG2 and HCCLM3 cells;ETV6 knockdown increased the p H and acidity of HepG2 and HCCLM3 medium;5.ETV6 overexpression reduced ROS in HepG2 and HCCLM3 cells by 33.5%(P=0.0001)and 28.8%(P=0.0020),respectively;ETV6 knockdown increased ROS by 23.9%(P=0.0037)and 51.5%(P=0.0010),respectively.6.ETV6 overexpression enhanced positive glycogen staining in HepG2 and HCCLM3 cells 1),Increasing number of magenta glycogen particles,are diffusely distributed in the cytoplasm of the cells,2)Glycogen content increased by 48.1%(P=0.0003)and 36.7%(P=0.0004),respectively,3)GCS activity increased by 62.6%(P=0.0001)and 51.4%(P=0.0005),respectively,4)GPa activity increased by 18.7%(P=0.0159)and 27.6%(P=0.0010),respectively;ETV6 knockdown weakened HepG2 and HCCLM3 cells 1),Purple red particles of light color,depletion in numbers,2)Glycogen content decreased by 43.4%(P=0.0044)and 38.5%(P=0.0012),respectively,3)GCS activity decreased by 35.8%(P=0.0003),34.9%(P=0.0003),respectively,4)The GPa activity was reduced by 21.8%(P=0.0022)and 14.9%(P=0.0066),respectively.7.ETV6 overexpression increased 1)GLUT1 expression in HepG2 and HCCLM3 cells by 43.1%(P=0.0067)and 51.2%(P=0.0426),respectively,2)HK2 expression was increased by 34.6%(P=0.0278)and 40.0%(P=0.0103),respectively,3)The expression of PFK1 was increased by 38.6%(P=0.0096)and 42.6%(P=0.0165),respectively,4)The expression of PKM2 was increased by 189.4%(P=0.0150)and 48.4%(P=0.0007),respectively,5)The expression of p-GSK3β was increased by 40.5%(P=0.0079)and 51.7%(P=0.0102),respectively,6)GSK3β expression decreased by 25.8%(P=0.0136)and 67.0%(P=0.0002),respectively;ETV6 knockdown reduced HepG2 and HCCLM3 cells 1)GLUT1 expression by 44.2%(P=0.0049),26.0%(P=0.0077),respectively,2)HK2 expression was decreased by 28.3%(P=0.0064),23.8%(P=0.0016),respectively,3)The expression of PFK1 was decreased by 66.6%(P=0.0037),21.8%(P=0.0038),respectively,4)The expression of PKM2 was decreased by 59.4%(P=0.0049),37.3%(P=0.0047),respectively,5)The expression of p-GSK3β was decreased by 40.8%(P=0.0002),33.2%(P=0.0287),respectively,6)GSK3β expression increased by 28.6%(P=0.0312)and 31.4%(P=0.0043),respectively.8.ETV6 overexpression increased 1)PI3K110 expression in HepG2 and HCCLM3 cells by 32.0%(P=0.0239)and 28.8%(P=0.0355),respectively,2)PI3K85 increased by 30.8%(P=0.0022)and 40.7%(P=0.0030),respectively,3)p-AKT increased by 36.0%(P=0.0006)and 36.3%(P=0.0002),No change in the AKT level;accordingly,After the knockdown of ETV6,1)PI3K110 expression in HepG2 and HCCLM3 cells decreased by 44.2%(P=0.0049)and 26.0%(P=0.0077),respectively,2)The PI3K85 level was decreased by 28.3%(P=0.0064)and 23.8%(P=0.0016),respectively,3)The p-AKT levels were decreased by 66.6%(P=0.0037)and 21.8%(P=0.0038),respectively,No change in AKT expression level.Conclusions:1.Changes in ETV6 expression levels were positively correlated with the aerobic glycolytic capacity of hepatoma cells;2.The ETV6 expression level was positively correlated with the glycogen metabolism capacity of hepatoma cells;3.ETV6 regulates cell glucose metabolism in hepatoma cells through the PI3K/AKT pathway.