| Hepatocellular carcinoma(HCC)is the most common primary liver cancer and the fifth most common cancer in the world.Due to limited treatment and poor prognosis,HCC patients have a high mortality rate.The most common causes of HCC are viral(chronic hepatitis B and C)infection,excessive alcohol consumption and aflatoxin infection,metabolic abnormalities(diabetes mellitus and nonalcoholic fatty liver disease),and immune factors(autoimmune hepatitis and primary biliary cirrhosis).Among them,abnormal expression of proto-oncogenes directly leads to the occurrence of liver cancer or plays a role of promoting cancer in the progression of liver cancer.Testicular specific protein 50(TSP50)is a testicular specific gene and a newly discovered proto-oncogene.TSP50 promotes the migration,invasion,and adhesion of various cancer cells,including breast cancer,colon cancer,and non-small cell lung cancer.At the same time,TSP50 also plays a regulatory role in the occurrence and development of liver cancer,but the specific mechanism has not been elaborated at present.Recent studies have found that the activation or inhibition of proto-oncogenes may cause abnormal changes in hepatocyte metabolism,which greatly promote the occurrence of hepatocellular carcinoma.Metabolic changes provide a more suitable environment for tumor growth,proliferation,and survival,because metabolic changes provide the right conditions for rapid proliferation of cancer cells,such as increased energy production,increased macromolecular biosynthesis,and maintenance of REDOX balance.Therefore,we speculated whether TSP50 regulates the occurrence of HCC by regulating the metabolic reprogramming in HCC cells.Based on the above theory,this study intends to investigate whether the TSP50 involves in the change of liver cell metabolism,and to explore TSP50 participates in metabolic reprogramming mechanisms.Then we hope to provide new ideas for the treatment of liver cancer.1.TSP50 regulates proliferation and metabolism reprogramming in liver cells1.1 TSP50 is highly expressed in liver cancer cells and promotes cell proliferationTSP50 is a key oncogene,which is highly expressed in multiple cancer cells.In present study,the protein expression of TSP50 is detected in L02,SMMC-7721,Huh7,Hep G2 and Bel-7402 cells.Result showed that TSP50 is higher expressed in hepatic carcinoma cells,especially in Huh2 cells and Bel-7402 cells.In order to further explore the function of TSP50 in hepatocyte,overexpression vector p EGFP-TSP50 or pc DNA3.1-TSP50 was constructed and transfected into L02 cells,and gene silencing vector sh TSP50 was transfected into Huh7 cells and Bel-7402 cells.Then,MTT and Brd U assay showed that overexpression of TSP50 promote the proliferation of L02 cells.Meanwhile,sh TSP50 inhibit the proliferation of Huh7 cells and Bel-7402 cells,indicating that TSP50 is a key gene affecting the proliferation of hepatocyte.1.2 TSP50 regulates metabolism reprogramming in hepatocyte and hepatoma carcinoma cellsTo investigate whether TSP50 promotes proliferation of hepatoma cells accompanied by changes in metabolic reprogramming.Overexpression of TSP50 reduce the ratio of NADP+/NADPH,while promote the synthesis of triglycerides and cholesterol.In addition,q PCR detection showed that overexpression of TSP50 significantly increase ACC,FAS,Fatp5 and Apo B in L02 cells,indicating that overexpression of TSP50 in L02 cells change the metabolic process in L02 cells.Since TSP50 is highly expressed in liver cancer cells,we transfected sh TSP50 vector into Huh7 cells and Bel-7402 cells.After detection,we found that sh TSP50 increase the NADP+/NADPH ratio,but inhibit triglyceride and cholesterol synthesis.Moreover,sh TSP50 significantly inhibit the expression of ACC,FAS and CD36,indicating that TSP50 regulate the changes in the proliferation ability and metabolism of HCC cells.2.TSP50-mediated metabolism changes regulate the proliferation of HCC cells2.1 TSP50 binds directly to G6 PD and they co-localize in the cytoplasmNext,we sought to determine the mechanisms by which TSP50 regulates the glucolipid metabolism and cells proliferation.Co-IP-MS was used for screening of proteins binding to TSP50.Multiple proteins can interact with TSP50,including G6 PD.Glucose-6-phosphate dehydrogenase(G6PD),the rate-limiting enzyme of the PPP,is elevated in HCC and contributes to tumor growth.Hence,we further verified the endogenous combination of TSP50 and G6 PD in Huh7 cells and Bel7402 cells.More importantly,co-immunoprecipitation experiments showed that endogenous TSP50 binds to endogenous G6 PD protein in Huh7 cells and Bel-7402 cells.In addition,Meanwhile,the GST-pull down experiment verified that GST-TSP50 and Flag-G6 PD can be directly combined in vitro.Importantly,their interaction could co-locate in the cytoplasm of Huh7 cells and Bel-7402 cells.Cytoplasm is a key location for the pentose phosphate pathway and tend to preferentially export more sugar metabolism-associated proteins via exosomes.2.2 TSP50 inhibits G6 PD acetylation and promotes G6 PD activityTo identify the expression and activity of G6 PD in L02 cells,Huh7 cells and Bel-7402 cells,western blot and glutaraldehyde cross-linking were used for analysis.Compare with L02 cells,G6 PD are higher expressed in Huh7 cells and Bel-7402 cells.In addition,cross-linking result showed that dimer and tetramer are the main forms in Huh7 cells and Bel-7402 cells.Consistently,G6 PD activity are higher in hepatoma cell lines.To further explore the impact of TSP50 on G6 PD,pc DNA3.1-TSP50 vector transfection led to increased expression of G6 PD in L02 cells.Simultaneously,the expression of G6 PD is significantly reduced in Huh7 cells and Bel-7402 cells after sh TSP50 treatment.Cross-linking was again verified that TSP50 promotes the formation of G6 PD dimers and tetramers and promotes the active form of G6 PD.To further investigate the mechanism,samples were collected after immunoprecipitation.The effect of TSP50 on G6 PD protein modification was verified,including acetylation,phosphorylation and O-glycosylation.Overexpression of TSP50 inhibit the acetylation of G6 PD.But TSP50 has little effect on p-Tyr,p-Thr,p-Ser and O-glycosylation.The above results indicated that TSP50 may regulate its activity by mediating G6 PD acetylation.Continuously,co-immunoprecipitation demonstrated that TSP50 can inhibit the binding of G6 PD protein to acetylase KAT9 and promote its binding to deacetylase SIRT2.In summary,TSP50 mediates its role in hepatocyte by promoting G6 PD expression and increasing enzyme activity,thus changing metabolic pathways in hepatocyte.2.3 TSP50 regulates acetylation of G6 PD K171 siteTo explore the G6 PD acetylation site affected by TSP50,pc DNA3.1-flag-G6 PD,G6PD K89 Q,G6PD K171 Q,G6PD K386 Q,G6PD K403 Q,G6PD K432 Q,G6PD K497 Q and G6 PD K514Q vectors using mutation kit were constructed for verification.G6 PD protein was purified by immunoprecipitation using anti-Flag antibody.Then each vector was transfected into L02 cells,and the G6 PD activity was detected after immunoprecipitation.It was found that the G6 PD enzyme activity in the transfected G6 PD K171Q,G6 PD K497Q and G6 PD K514Q groups are significantly lower than that in the control group,indicating that sites K171,K497 and K514 in HCC cells are the key sites affecting the G6 PD enzyme activity.In order to further determine the targeted regulation sites of TSP50,changes in G6 PD enzyme activity were detected after co-transfection of pc DNA3.1-TSP50 with Flag-G6 PD,G6PD K171 Q,G6PD K403 Q,G6PD K497 Q and G6 PD K514Q.The results showed that there is no significant difference in cells that TSP50 co-transfected with G6 PD K171Q,revealing that TSP50 regulates acetylation of G6 PD K171 sites.For further study whether TSP50 participate in the acetylation of G6 PD by affecting its binding with KAT9 and SIRT2,co-immunoprecipitation analyzed the effect of overexpression of TSP50 on the binding ability of G6 PD with acetylase and deacetylase.The results showed that overexpression of TSP50 promotes G6 PD binding to deacetylase SIRT2 and inhibits its binding to acetylase KAT9.It was partly explained that TSP50 inhibits the acetylation level of G6 PD K171 by regulating the binding of G6 PD with acetylase.In order to more accurately verify the regulation of G6 PD acetylation site by TSP50,we designed a peptide corresponding to G6 PD K171 site and immunized white rabbits.Finally,the acetylation antibody an-G6 PD K171 site was harvested.Then pc DNA3.1-TSP50 transfect into L02 cells.And sh TSP50 transfect into Huh7 and Bel-7402 cells,then the acetylation of G6 PD K171 was detected.The results again verified that TSP50 partially inhibit the acetylation of G6 PD K171 site.2.4 TSP50 mediated G6 PD activity changes regulate metabolism and alter cells proliferationTo confirm that G6 PD K171 acetylation is involved in hepatoma cells proliferation and metabolism,TSP50 co-transfected with Flag-G6 PD,Flag-G6 PD K171Q and FlagG6 PD K403Q into L02,Huh7 and Bel-7402 cells,respectively.MTT and Brd U assay showed that the cell proliferation abilities are significantly inhibited in group of TSP50 and Flag-G6 PD K171Q co-transfection.In addition,after co-transfection with pc DNA3.1-TSP50 and G6 PD K171Q,the formation of intracellular triglyceride and cholesterol content are significantly lower than that of the group co-transfected with pc DNA3.1-TSP50 and wild-type G6 PD.These results indicated that the acetylation of G6 PD 171 site directly affect the lipid synthesis and cell proliferation in L02 cells,Huh7 cells and Bel-7402 cells.At the same time,the relative expressions of ACC,FAS,Fatp2 and Fatp5 in the TSP50 and G6 PD K171Q co-transfected group are also significantly lower than those in the control group,suggesting that G6 PD acetylation regulate the expression of lipid-related genes,which may partly explain the TSP50-mediated G6 PD enzyme activity regulating the metabolic reprogramming in liver cancer cells.2.5 TSP50 regulates NADP+/NADPH ratio and G6 PD enzyme activity to affect metabolism signaling pathways and to alter cells proliferationSince TSP50 regulates the ratio of NADP+/NADPH in liver cancer cells,proliferation was detected by gradient addition of NADP+.And it was found that NADP+ inhibit the proliferation levels of Huh7 and Bel-7402 cells in a dose-dependent manner.This also explains the phenomenon that TSP50 changes metabolic reprogramming to regulate the proliferation ability of HCC cells in another direction.In addition,since TSP50 directly binds to G6 PD and regulates the acetylation of G6 PD K171 site,thus affecting the enzyme activity of G6 PD,Huh7 cell were treated with RRX-1,an inhibitor of G6 PD enzyme activity.The result shown that G6 PD enzyme activity changes affect cellular metabolism and proliferation of liver cancer gene enrichment.In addition,liver cancer oncogene and nonalcoholic fatty hepatitis genes are enrichment.Moreover,it will lead to the enrichment of the signal pathways of lipid metabolism,glucose metabolism and amino acid metabolism in liver cancer cells,which further indicates that the change of G6 PD enzyme activity caused by G6 PD acetylation is a key factor mediating the occurrence of liver cancer diseases.2.6 TSP50 regulates G6 PD K171 acetylation to promote xenograft tumor formationIn order to explore the effect of TSP50 and acetylation of G6 PD K171 on tumor formation and growth in vivo,we constructed a xenograft tumor model using L02 cells.First,p LVX-Ac GFP-N1,p LVX-Ac GFP-TSP50,p LVX-Ac GFP-G6 PD,p LVX-Ac GFPG6 PD K171Q lentivirus vectors were packed.And then the cells were transfected according to the group p LVX-Ac GFP-N1,p LVX-Ac GFP-TSP50,p LVX-Ac GFPTSP50+p LVX-Ac GFP-G6 PD,p LVX-Ac GFP-TSP50+p LVX-Ac GFPG6 PD K171Q,respectively.The cells were infected into the back of nude mice.After feeding,it was found that p LVX-Ac GFP-N1 group cannot lead to tumors formation,while the other three groups form tumors.Moreover,tumor weight and volume measurements showed that the weight and volume of p LVX-Ac GFP-TSP50 and p LVXAc GFP-G6 PD co-transfected groups are significantly higher than those of the other two groups.To further verify that acetylation of TSP50 and G6 PD K171 regulates lipid metabolism in xenograft tumors.After detection,the content of triglyceride and cholesterol in the tumor tissues of the p LVX-Ac GFP-TSP50 and p LVX-Ac GFP-G6 PD co-transfected group are significantly higher than that of the other two groups.In addition,the expression levels of ACC,FAS,Fatp2,Fatp5 and CD36 genes are highest expressed in the wild-type co-transfection group.The above results further elucidate the important role of TSP50 and G6 PD in tumor formation and the significance in regulating lipid metabolism during tumor formation.These results underscore the importance of TSP50-regulated acetylation of G6 PD in the pathogenesis and progression of tumors.3.The TSP50 and G6 PD expression are significantly correlated with HCCpatients survivingIn order to understand whether TSP50 and G6 PD are related to cancer in clinical patients,the information of liver cancer patients was collected and analyzed from TCGA database.It was found that the expression levels of TSP50 and G6 PD in liver cancer patients are significantly higher than that in normal liver tissues.In addition,correlation analysis between the expression levels of TSP50 and G6 PD after clinical staging of liver cancer showed that the expression levels of TSP50 and G6 PD are positively correlated with the clinical staging.Furthermore,364 samples with higher expression and lower expression of TSP50 and G6 PD were selected from liver cancer tissues for survival analysis.The results showed that there is significant negative correlation between the expression levels of these two proteins with the survival time of patients.This further highlights the role of TSP50 and G6 PD in the development and progression of HCC.In present,we demonstrated that TSP50 changes the lipid metabolism and signal pathway in hepatocytes by regulating the acetylation of G6 PD,thus affecting the proliferation of hepatocytes and the formation of xenograft tumors.The relationship between reprogramming of liver cancer metabolic changes and the occurrence of liver cancer was analyzed in detail,providing a new perspective for the treatment of liver cancer. |
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