The Role And Molecular Mechanism Of ATP6V1G1 In Lipid Metabolism Of Hepatocellular Carcinoma

Aims and significance Hepatocellular carcinoma(HCC)is a primary invasive tumor of the liver with a high incidence and poor prognosis.Its occurrence and progression are regulated by multiple genes and involve multiple factors.Most of the metabolic processes in the human body,including glucose metabolism,amino acid metabolism,and lipid metabolism,occur in the liver.Among them,the lipid metabolism of liver cancer patients can provide and reserve the energy required for the proliferation of rapidly growing cancer cells,and lipid accumulation has become an important feature of HCC.In recent years,studies have confirmed that targeting V-ATPase can inhibit tumor growth by interfering with cancer cell lipid metabolism.We previously detected that ATP6V1G1 was highly expressed in HCC tissues.Exogenous overexpression of ATP6V1G1 in HCC cells can promote cell proliferation,invasion and metastasis.This study found that knocking down the key subunit ATP6V1G1 of V-ATPase can reduce the accumulation of lipid droplets in liver cancer cells and significantly inhibit tumor growth,which revealed a new regulation mechanism of lipid metabolism in HCC,to provide new ideas and evidence support for the treatment plan targeting lipid metabolism.Methods Lentiviral transfection technology was used to overexpress and silence ATP6V1G1.Based on phosphorylation proteomics and bioinformatics analysis,it was found that ATP6V1G1 is involved in various metabolic pathways including lipid metabolism in HCC cells.The conclusions of the phosphorylated proteome were validated using BODIPY? 493/503 staining,Oil Red O staining,intracellular triglyceride,and cholesterol detection.The ATPase kit and lysosomal probe were used to detect the effects of ATP6V1G1 on total ATPase and V-ATPase activities,and qRT-PCR was used to detect the changes of lysosomal synthesis-related genes.Besides,the V-ATPase inhibitor bafilomycin A1 was used to inhibit the activity of V-ATPase in liver cancer cells,and its effect on lipids was observed.Further qRT-PCR and Western blot techniques were used to detect the key enzymes that ATP6V1G1 might regulate lipid metabolism.It was identified that ATP6V1G1 may affect lipid metabolism through the key enzyme SCD1 of unsaturated fatty acid synthesis.Then,the SCD1 product oleic acid was used to treat ATP6V1G1-silenced HCC cells,and oil red O staining,CCK8 assay,and PI staining were used to observe the changes of lipid droplet formation,proliferation,and death of cells.The decrease of ATP level in liver cancer cells after ATP6V1G1 silencing and the pathway analysis results of phosphorylation proteomics all suggest that the energy-sensing pathway AMPK may undergo corresponding changes.Western blot was used to verify the changes of AMPK subunits and phosphorylation levels.ATP6V1G1 silenced cells were treated with AMPK pathway inhibitors and activators to observe the biological function of liver cancer cells and the changes of AMPK phosphorylation level and target protein SCD1.In addition,this study analyzed the relationship between ATP6V1G1 and lipogenic enzyme SCD1 in liver cancer patients by the visualizing bioinformatics website GEPIA2.The tissue specimens,survival and clinical data of 50 HCC patients were collected,and the relative expressions of ATP6V1G1 and SCD1 were detected by immunohistochemical staining,and their effects on the overall survival time and recurrence time of HCC patients were analyzed.Finally,univariate and multivariate Cox regression models were used to analyze and screen out the statistically significant high-risk factors for liver cancer death and recurrence.These variables and corresponding regression coefficients were used to construct the overall prognosis risk score model and recurrence score risk model of HCC,respectively.Results The results of qRT-PCR and Western blot showed that the ATP6V1G1 overexpressed Hep G2 and Huh7 cell lines,as well as the ATP6V1G1 silenced Hep3 B and Huh7 cell lines were successfully constructed.Based on the phosphorylation proteomics technology,the effect of ATP6V1G1 upregulation on the function of HCC cells was analyzed,and the number of phosphorylated proteins,peptides,and sites was identified as 1873,4747,and 5897,respectively.Western blot results showed that in the ATP6V1G1 overexpression group,p-RPS6(Ser235)and p-SQSTM 1(Ser272)were up-regulated,while p-PDPK1(Ser241)and p-EEF2(Ser57)were down-regulated,which consistent with the phosphoproteomic results.Bioinformatics analysis of phosphoproteomic revealed that the main enriched differentially phosphorylated proteins and the main KEGG pathways related to the occurrence and development of HCC included "necroptosis","apoptosis","HIF-1 signaling pathway","m TOR signaling pathway","AMPK signaling pathway","PI3K-Akt signaling pathway",etc.The results of KEGG analysis also indicated that changes in the expression of ATP6V1G1 affected multiple metabolic pathways of liver cancer cells,including amino acid metabolism,carbohydrate metabolism,lipid metabolism,and nucleotide metabolism.It has been confirmed that ATP6V1G1 is an important subunit for maintaining V-ATPase activity and targeting V-ATPase can inhibit tumor growth by interfering with cancer cell lipid metabolism.Through related tests,we confirmed that ATP6V1G1 silencing reduced total ATPase activity,V-ATPase activity,lipid droplet synthesis,and intracellular triglycerides.And lysosome synthesis related-genes LAMP1,LAMP2,CD63,and TRPML1 were increased or decreased in different degrees,suggesting that synthesis and function of lysosome were disorders.The small-molecule inhibitor bafilomycin A1 inhibited the expression of lipid droplets and triglycerides in liver cancer cells while inhibiting the activity of V-ATPase.It was further found that the silencing of ATP6V1G1 promotes the disordered expression of liposynthase ACC1,FASN,SCD1,and lipolytic enzymes ATGL,MGLL,and LIPE,but the changes are not uniform in different liver cancer cells.Only the key enzyme SCD1 for unsaturated fatty acid synthesis was decreased in both Hep3 B and Huh7 cells.And the rescue experiment results show that the SCD1 product oleic acid could reverse the decrease in lipid droplets,the decrease in cell proliferation,and the increase in cell death caused by ATP6V1G1 silencing.Down-regulation of the lipid metabolism pathway acted by ATP6V1G1 reduced the overall ATP level in HCC cells,and the results of phosphorylation proteomics pointed to the effect of ATP6V1G1 on the AMPK pathway related to energy metabolism.Using Western blot technology,it was proved that ATP6V1G1 increased the levels of AMPK catalytic subunits α1 and α2,and decreased the levels of regulatory subunits β1,β2,γ1,and γ3;and increased the phosphorylation level of AMPKα.The decreased proliferation and increased cell death of Hep3 B and Huh7 hepatoma cells induced by ATP6V1G1 silencing were significantly reversed by the use of the AMPK pathway inhibitor Compound C.At the same time,it was accompanied by a decrease in the phosphorylation level of AMPK and an increase in the expression of the downstream target protein SCD1 in hepatoma cells in a concentration-dependent manner of Compound C.Conversely,treatment of the AMPK pathway activator AICAR significantly enhanced the decreased proliferation and increased cell death of Hep3 B and Huh7 hepatoma cells caused by ATP6V1G1 silencing.At the same time,it was accompanied by increased AMPK phosphorylation and decreased SCD1 expression in a concentration-dependent manner with AICAR.Bioinformatics analysis found a significant positive correlation between the expression of ATP6V1G1 and SCD1 in tumor tissues of patients with liver cancer.Both high expressions of ATP6V1G1 and SCD1 are associated with lower survival rates and higher recurrence rates of liver cancer patients.We screened out four variables,including Child-pugh grade,maximum tumor diameter,ATP6V1G1,and SCD1 to construct an overall risk score model(Overall Risk-Score = 1.467 × Child-pugh grade + 0.177 × maximum tumor diameter + 3.422 × ATP6V1G1 + 2.291 × SCD1);Three variables of child-pugh class,ATP6V1G1 and SCD1 were included in the recurrence risk score model(Recurrence Risk-Score = 2.795 × child-pugh class + 1.147 × ATP6V1G1 +0.962 × SCD1),these two models predicted the overall prognosis of liver cancer patients or The AUCs for recurrence were 0.974 and 0.804,respectively,and the HRs were 12.43(95%CI,5.47-28.26)and 4.17(95%CI,1.91-9.10),respectively.Conclusion This study revealed that ATP6V1G1 affects the activity of V-ATPase in liver cancer cells and positively regulates liver lipid metabolism.SCD1 is a key condition for ATP6V1G1 deficiency-mediated reduction of lipogenesis and is regulated by AMPK phosphorylation.Down-regulation of the lipid metabolism pathway acted by ATP6V1G1 inhibited HCC cell growth by reducing SCD1 expression and enhancing AMPK phosphorylation.The expression analysis of ATP6V1G1 and SCD1 can evaluate the survival and prognosis of HCC patients,and the risk assessment model constructed by using the two and clinical data can more accurately predict the prognosis of HCC.The novel mechanism by which ATP6V1G1 regulates SCD1-mediated lipid metabolism is expected to become a new target for HCC therapy.