Mechanisms Of ASNS Against Apoptosis In Hepatocellular Carcinoma During Glutamine Starvation

Background: Primary liver cancer is one of the most common malignancies in the world,the fourth leading cause of cancer-related deaths,and the second leading cause of cancer-related deaths in China.There are two main types of primary liver cancer-hepatocellular carcinoma(HCC)and intrahepatic cholangiocarcinoma(ICC).The former accounts for more than 80% of primary liver cancer cases worldwide.Glutamine,as an important nutrient for tumor cell growth and metabolism,provides sufficient carbon and nitrogen sources for their growth and development.It provides energy for cell growth by participating in the tricarboxylic acid cycle and is also involved in the formation of reduced glutathione to maintain intracellular redox homeostasis.Due to the special metabolic pattern of tumor cells,their glutamine requirement is much higher than that of normal cells,therefore,the ability of tumor cells to cope with glutamine deficiency is extremely important to them.Because of the special metabolic laws of tumor cells,their glutamine requirements are much greater than those of normal cells,so the ability of tumor cells to cope with glutamine deficiency is extremely important to them.In order to cope with the unfavorable situation of glutamine deficiency,hepatocellular carcinoma cells must meet the material and energy requirements for their survival and proliferation by adjusting their own status to provide for their survival and development.Asparagine Synthetase(ASNS)transcripts encode asparagine synthetase,which converts glutamine and aspartate to asparagine and glutamate in the cytoplasm of tumor cells.Most studies on ASNS at home and abroad have focused on the effect of its ability to synthesize asparagine on tumor cells,but its relationship with glutamine metabolism in hepatocellular carcinoma cells is unclear.In addition,the role and mechanism of ASNS in hepatocellular carcinoma cells facing glutamine starvation microenvironment are not yet clear.Objective: To investigate the specific functions and molecular mechanisms played by ASNS under glutamine starvation conditions in hepatocellular carcinoma cells and their physiological significance,and to provide new ideas for future clinical target therapy for hepatocellular carcinoma patients.Methods: Western blot and qRT-PCR were used to detect the levels of relevant proteins as well as mRNA expression levels under glutamine starvation conditions;using specific ASNS shRNA,lentiviral packaging and transfection were used to establishASNS knockdown tumor cell lines,and the effect of ASNS on cell proliferation was detected by cell growth count and plate cloning assay;after knockdown of ASNS,the cell phenotype was observed under The effect of ASNS on cell proliferation was determined by cell growth count and plate cloning assay;the cell phenotype was observed under microscope after knockdown of ASNS,and the effect of ASNS on cell death was determined by Western blot to detect proteins associated with cell death under glutamine starvation conditions;the effect of ASNS on cell apoptosis was detected by flow cytometry using Annexin V/PI apoptosis staining of HepG2 cells under different nutritional conditions after knockdown of ASNS;the effect of ASNS on cell apoptosis was detected by flow cytometry using γ H2 AX antibody staining to further verify the effect of ASNS on apoptosis by immunofluorescence;knockdown of ASNS in HepG2 cells followed by Western blot and qRT-PCR to detect changes in apoptosis-associated proteins under normal nutritional conditions and glutamine starvation;immunoblotting with ubiquitin-specific antibodies to detect ubiquitination levels of STAT3;and Immunoprecipitation assay was performed to assess the correlation between ASNS and STAT3.Results: Under glutamine starvation conditions,ASNS is significantly elevated by transcriptional regulation;ASNS inhibits apoptosis and promotes survival of hepatocellular carcinoma cells under glutamine deficient conditions by promoting the expression of apoptosis suppressor protein Bcl-2 and inhibiting the expression of apoptosis promoting protein Bax;further mechanistic studies revealed that ASNS promotes the expression of STAT3,which binds to STAT3 and inhibits STAT3 acts as a transcription factor for Bcl-2 and Bax,and an increase in its protein content causes an increase in Bcl-2protein content and a decrease in Bax protein content,thereby inhibiting the endogenous apoptotic pathway.survival in the glutamine-deprived microenvironment.Conclusion: ASNS expression was upregulated in hepatocellular carcinoma cells facing glutamine deprivation microenvironment,and promoted cancer cell survival by reducing ubiquitinated degradation of STAT3,which in turn inhibited the onset of endogenous apoptosis.This study reveals the important role of ASNS in promoting cell survival when hepatocellular carcinoma cells are exposed to glutamine starvation,and this finding can help us better understand the metabolic reprogramming mechanism of hepatocellular carcinoma cells and contribute to future clinical targeting therapy for hepatocellular carcinoma patients.