The Mechanism Of SIRT1 Regulating PKM2 Deacetylation To Inhibit Protein Stability And The Effects In Metabolism Of Gastric Cancer

Multiple studies have shown that reprogramming of energy metabolism is one of the markers of tumor cells,and also a key factor affecting tumor occurrence and development.Under the condition of sufficient oxygen,tumor cells are tend to convert glucose metabolism into lactic acid.This phenomenon is known as aerobic glycolysis,or the "Warburg effect".In recent years,a considerable number of researchers have focused on exploring the carcinogenic effects of aerobic glycolysis.Increased glucose uptake,faster consumption,and lactic acid release rate are notable features of cellular aerobic glycolysis.The glycolysis process is significantly increased in tumor cells,which enables tumor cells to obtain large amounts of ATP through rapid use of glucose,and then through pentose phosphate pathway,produce rich synthetic raw materials,such as nucleotides,lipids and non-essential amino acids,and accelerate the uncontrolled proliferation of tumor cells.In addition,lactic acid derived from glycolysis pathway is an important substance that causes changes in tumor microenvironment.Acidic tumor microenvironment affects tumor metastasis and immune response,and is involved in tumor progression.Therefore,glycolysis may be a promising target in the treatment of cancer patients.However,due to the complexity of Warburg effect,its molecular mechanism in gastric cancer has not been fully elucidated.1、PKM2 is highly expressed in GC and negatively correlated with patient survivalFirstly,UALCAN database was used to predict the transcription level of PKM2 in GC.The results showed that the mRNA level of PKM2 in TCGA-STAD tissue was significantly increased compared with that in paracancer normal tissue.We further predicted the relationship between PKM2 and clinical case characteristics of the TCGA-STAD sample,including patient sex,age,tumor stage and grade.The results showed that the expression level of PKM2 in tumor tissue of GC patients was significantly related to tumor grade.According to the PKM2 mRNA expression level,GC patients were divided into high and low PKM2 expression groups.Kaplan-Meier survival curve was used in GEPIA database to analyze whether high/low PKM2 expression was associated with the survival of GC patients.The results showed that patients with high PKM2 expression had shorter relapse-free survival(RFS).Therefore,PKM2 may be a possible prognostic marker for GC.Further,by detecting the expression of PKM2 in clinical patients with gastric cancer,we found that the mRNA and protein levels of PKM2 were significantly higher than those of normal control group.2、Correlation between PKM2 regulation of glucose metabolism and its effect on cell proliferationFurther,to investigate the biological function of PKM2,expression levels of PKM2 were detected in normal gastric mucosal epithelial cells(GES-1)and gastric cancer cells(AGS,SGC7901,MKN45,and MKN72).The results showed that PKM2 expression level was low in GES-1 cells,but high in gastric cancer cell lines.Normal GES-1 cells,AGS and SGC7901 gastric cancer cells were selected for further experiments.To further investigate the function of PKM2,we constructed overexpression and knockdown vectors oe-PKM2 and si-PKM2 and transferred them into AGS and SG7901 cells.The effect of PKM2 on cell proliferation was analyzed by CCK-8 and clonal formation assay,and Transwell assay assessed cell invasion and migration.The data indicated that PKM2 significantly promoted the proliferation,invasion,and migration of GC cells.In order to determine whether PKM2 affects the glucose metabolism of tumor cells,we used relevant kits to detect the change levels of glycolytis-related indicators ATP production,glucose uptake,and lactic acid production).The results showed that PKM2 promoted the incidence of aerobic glycolysis.3、SIRT1 deacetylated PKM2 K280 siteWe attempted to investigate whether the acetylation of PKM2 is regulated by SIRT1.In general,PKM2 is usually acetylated at the K335 lysine and K280 lysine sites.Therefore,in order to simulate the continuous non-acetylated PKM2,we constructed the K335R and K280R mutant plasmids SIRT1 overexpressed plasmids transfected into K335R and K280R cells were purified PKM2 protein by immunoprecipitation method.western blot analysis showed that SIRT1 was not related to the activity of PKM2 and K280R After overexpression or knockdown of SIRT1,the acetylation PKM2 K280 was measured using specific antibody.The results showed that SIRT1 significantly reduced the acetylation level of PKM2 K280 Taken together,these results suggest that SIRT1 causes deacetylation at the K280 site of PKM2.4、SIRT1 regulates PKM2 acetylation to inhibit tumor formationFinally,we established a xenotransplantation mouse model and analyzed whether SIRT1/PKM2 regulated tumor formation and growth(n=6/group).AGS cells stably expressing SIRT1,PKM2 WT,SIRT1+PKM2 WT were prepared by lentivirus infection,and then 5 × 107 AGS cells were subcutaneously injected into the back of female nude mice.The results showed that four weeks after injection,all nude mice in the injection group formed tumors.Compared with the control group,the tumor size and weight of nude mice in PKM2 WT group were significantly increased,and the level of glycolysis was significantly increased.In addition,compared with PKM2 WT group,SIRT1+PKM2 WT group significantly inhibited tumor volume and reduced tumor weight in mice overexpressing PKM2.In addition,the glycolysis level of tumor tissue transfected with SIRT1+PKM2 WT was significantly reduced.