| Background:Primary liver cancer can be divided into three main pathological types: hepatocellular carcinoma(HCC),intrahepatic cholangiocarcinoma(ICC),and HCC-ICC mixed type.The three types are quite different in pathogenesis,biological behavior,histomorphology,treatment methods,and prognosis.Among them,the incident rate of HCC reaches 85% ~90%of the population.Therefore,selecting HCC as a type of liver cancer has high clinical significance.With the continuous development of technology and the in-depth study of the pathogenesis of liver cancer,new understanding of metabolic reprogramming occurred in the microenvironment of liver cancer has been gained,which also triggered new thinking on the treatment of primary liver cancer.Metabolic reprogramming is one of the important signs of the occurrence and development of cancer.Through metabolic reprogramming,tumor cells can maintain their own energy supply and maintain the malignant transformation and proliferation of tumor cells in the face of hypoxia and low nutrition in the microenvironment of cells.Warburg effect is also a kind of metabolic reprogramming,which is more common in cancer cells.When Warburg effect occurs in cancer cells,the cells can still rely on aerobic glycolysis to generate energy when the oxygen is sufficient.Due to the low energy conversion efficiency of glycolysis,cancer cells absorb a large amount of glucose and form a high-lactic acid environment in the cells,resulting in the malignant phenotype of tumors.One of the reasons for Warburg effect is glutamine addiction in cancer cells.Glutamine addiction can lead to the enhancement of intracellular glycolysis pathway.Although glucose is one of the main sources of aerobic glycolysis,in the development of cancer cells,because glucose can not provide sufficient energy supply for cancer cells in time,cancer cells will choose more readily available glutamine to supplement energy.Because of the dependence of cancer cells on glutamine,the Warburg effect occurs in cancer cells,This enables cancer cells to carry out glycolysis to generate sufficient energy supply even when there is sufficient oxygen in the cells.In the aerobic glycolysis pathway of cancer cells,glutamine can be decomposed into pyruvic acid through glutaminase,and pyruvic acid can be directly converted into lactic acid,promoting the glycolysis pathway to generate energy.In normal cells,pyruvate produced in aerobic glycolysis pathway needs to be converted into acetyl coenzyme A through pyruvate dehydrogenase complex to participate in the tricarboxylic acid(TCA cycle)in cells.Glutamine is hydrolyzed by glutaminase for glutamylation,and the generated glutamic acid can be converted into α-Ketoglutaric acid(α-KG)and then promote the TCA cycle of cells.α-KG also has a certain compensatory effect on TCA cycle,which can promote the conversion of pyruvate to acetyl coenzyme A,maintain the normal TCA cycle of cells,and ensure the normal energy supply of cells.Glutinase can convert glutamine into different metabolites and provide sufficient energy supply for different cell states.When glutamine addiction occurs in cancer cells,the intracellular microenvironment changes,and glutamine decomposition promotes the transformation of glycolysis pathway.Glutamine decomposition has certain selectivity for glutaminase,which can be divided into kidney type glutaminase and liver type glutaminase according to different distribution positions.At present,two kinds of mammalian an glutaminase GAs have been identified: liver type(LGA or GLS2),kidney type(KGA)and glutaminase C(GAC,GLS).GLS and GLS2 present different distribution degrees to different tissues.In this study,we mainly focused on the relationship between the high expression of GLS in liver cancer cells and glutamine addiction and glycolysis pathway,the prognostic significance of the high expression of GLS in liver cancer tissues and the analysis of the correlation between GLS and immune cells,and the study of promoting the proliferation and migration of liver cancer cells.Research materials and methods:First,the expression map of GSE76427 dataset was downloaded from the database TCGA and the database GEO,and the differential gene expression analysis,GO/KEGG enrichment analysis,and survival relationship analysis were performed to analyze the prognostic significance of GLS single gene in liver cancer tissue and its correlation with immune cells.After establishing the optimal experimental concentration of glutamine-dependent hepatoma cells through experiments,it was proved that glutamine addiction promoted the growth and proliferation of hepatoma cell Hep G2 through cell count,CCK-8 experiment and cell cloning experiment,and it was verified by q RT-PCR experiment that hepatoma cells expressed GLS abnormally high in the environment of glutamine addiction.Then the optimal concentration of GLS inhibitor for hepatoma cells was determined through CCK-8 experiment to pave the way for the best results in subsequent experiments.Through cell cloning experiment,cell scratch experiment and Transwell experiment,it was proved that GLS can promote the growth,proliferation and migration of hepatoma cell Hep G2.The determination of lactic acid content proved that GLS promoted glycolysis of liver cancer cells.Then replenish different concentrations α-KG,different concentrations proved by CCK-8experiment α-KG affects the growth of hepatoma cells.It is proved by RT-PCR that different concentrations of α-KG affects the expression of GLS m RNA,and the detection of lactate content and hexokinase content proves that different concentrations of rehydration α-The influence of α-KG and BPTES on glycolysis pathway was verified by Western Blot experiment The effects of α-KG and BPTES on PI3K-AKT pathway and the expression of proteins related to the growth,proliferation and migration of hepatoma cells were verified.Research results:Through the verification of bioinformatics analysis,it was proved that the high expression of GLS was positively correlated with poor prognosis and immune infiltration of liver cancer tissue,and confirmed that the high expression of GLS had the role of malignant tumor marker in liver cancer cells.Through different experimental verifications,it was determined that 2m M glutamine was the optimal growth concentration for hepatoma cell dependence.Glutamine addiction could promote the growth and proliferation of hepatoma cells,and promote the high expression of GLS m RNA in hepatoma cells without significant effect on normal hepatocytes.It was determined that the optimal concentration of GLS inhibitor BPTES acting on hepatoma cell Hep G2 was 5 μM.The cell count experiment proved that inhibition of GLS can inhibit the growth of Hep G2,the CCK-8 experiment proved that inhibition of GLS can inhibit the activity of Hep G2,the cloning experiment proved that inhibition of GLS can inhibit the proliferation of Hep G2,the cell scratch experiment proved that inhibition of GLS can inhibit the migration of Hep G2,and the Transwell experiment proved that inhibition of GLS can inhibit the invasion of Hep G2,The determination of lactic acid content showed that inhibition of GLS could inhibit the glycolysis of Hep G2 cells.Then replenish different concentrations of α-KG,which is verified by CCK-8 experiment with different concentrations α-KG affects the activity of Hep G2 cells and selects 2m M and 10 m M α-The two key nodes of α-KG were verified by subsequent experiments.The expression of GLS m RNA at different concentrations was verified by q RT-PCR experiment,and the cloning experiment was continued to verify2 m M α-KG promotes the proliferation of Hep G2 cells,10 m M α-KG inhibits the proliferation of hepatoma cells;Verify2m M through cell scratch test α-KG promotes the migration of Hep G2 cells,10 m Mα-KG inhibits the migration of hepatoma cells;Verify 2m M through Transwell experiment α-KG promotes the invasion of Hep G2 cells,10 m M α-KG inhibits the invasion of hepatoma cells.Validate 2m M by detecting lactic acid content and hexokinase content α-KG promotes glycolysis of Hep G2 cells,10 m M α-KG inhibits glycolysis of hepatoma cells.Verified by Western Blot experiment,2m Mα-KG activates PI3K-AKT signal pathway in Hep G2,10 m M α-KG inhibits the PI3K-AKT signal pathway in hepatoma cells.Finally,Western Blot test was performed on the proliferation and migration related proteins of hepatoma cells to verify 2m M α-KG promotes the proliferation and migration of Hep G2 cells,10 m M α-KG inhibits the proliferation and migration of hepatoma cells.Analysis conclusions:1.The high expression of GLS is the key sign of glutamine-dependent addiction of liver cancer cells.It can promote the proliferation and migration of liver cancer cells by enhancing glutamine addiction.2.The high expression of GLS promotes the glycolysis pathway in hepatoma cells,and promotes the proliferation and migration of hepatoma cells.3.GLS expression is affected by α-The double regulatory effects of KG and BPTES can promote or inhibit the growth,proliferation and migration of liver cancer cells. |
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