Clinical Significance And Molecular Mechanism Of ATP6V0D2 In Hepatocellular Carcinoma

Purpose and significanceHCC(Hepatocellular carcinoma,HCC)is an invasive malignant tumor,which ranks first in tumor morbidity and mortality in the world,and it is also one of the most common malignant tumors in China.Guangxi is an area with high incidence of HCC,and its morbidity and mortality are much higher than the national level.Clinical research data show that the difficulty of early diagnosis and the high rate of recurrence and metastasis are the main reasons for the high mortality of patients with HCC.With the development of high-throughput gene detection technology and the continuous maturity and application of bioinformatics analysis methods,big data analysis was carried out by mining database to study the molecular mechanism of the occurrence and development of HCC and to find early diagnostic markers and prognostic target molecules of HCC,which provided a new idea for studying the pathological basis of HCC.Tumor microenvironment plays an important role in the formation and development of cancer,which determines the fate and prognosis of tumor cells.Acidic microenvironment is one of the important factors such as anti-apoptosis,invasion and spread,multiple drug resistance and promoting neovascularization of tumor cells.The biological characteristics of V-ATPase provide a specific PH environment for some biochemical reactions in vivo.Some subunits of V-ATPase are highly expressed in many tumors.At present,there are only a few reports about the abnormal expression of V-ATPase subunits in liver-associated cancer.The main purpose of this study is to mine the ATP6V0D2 gene expression data of HCC samples obtained from The Cancer Genome Atlas(TCGA)database,and analyze its clinical diagnosis,prognosis evaluation,biological function and related signal pathways in HCC,and then study its role in the occurrence and development of HCC through cell function experiments,and explore its molecular mechanism of promoting HCC formation.Research Methods1.The expression of ATP6V0D2 in HCC and its clinical significance were analyzed by TCGA database.(1).Download the expression dataset of HCC gene transcriptome in TCGA database through GDC,and extract the ATP6V0D2 expression data of HCC samples and normal control samples adjacent to cancer after sorting.(2).The R software was used to analyze the difference of ATP6V0D2 expression between HCC samples and adjacent normal control samples,HCC samples and paired adjacent normal control samples.(3).Download and sort out HCC clinical data from TCGA database through GDC.(4).The correlation analysis between the expression of ATP6V0D2 and the survival,clinical characteristics of HCC patients were performed via R software.(5).The independent prognostic value of ATP6V0D2 in HCC was analyzed by R software.(6).Receiver operating characteristic curve(ROC)was used to analyze the efficacy of ATP6V0D2 expression in the diagnosis of HCC.(7).Gene Set Enrichment Analysis was used to explore the different cell signaling Pathways between the high and low expression groups of ATP6V0D2 in HCC.(8).Multiple databases were used to analyze the correlation between ATP6V0D2 and infiltrating immune cells and Tumor Infiltrating Lymphocytes.2.Study on the effect of ATP6V0D2 on the Biological function of Hepatocellular carcinoma cells(1).Stable cell lines overexpressing/knockdown ATP6V0D2 gene(Hep-3B and Huh-7)were constructed using lentiviral vectors,and transfection efficiency was verified.(2).Cell proliferation was detected by CCK-8 test,cell invasion and migration were detected by Transwell test,apoptosis was detected by flow cytometry and ATPase activity was detected by enzyme-linked immunosorbent assay,which aim to study the effect of ATP6V0D2 on the biological function of hepatocellular carcinoma cells.(3).The mRNA expression of LC-3B,Beclin-1,AKT and mTOR in hepatocellular carcinoma cells and HCC samples was detected by real-time fluorescence quantitative polymerase chain reaction(RT-q PCR),and the expression of these molecular proteins was detected by Western blotting to explore the signal pathway of ATP6V0D2 affecting the biological function of hepatocellular carcinoma cells.Results1.The expression and clinical significance of ATP6V0D2 in HCC.(1).By sorting and analyzing the gene transcript group data of HCC samples in TCGA database,the expression of ATP6V0D2 in adjacent control samples and HCC samples was extracted.After statistical analysis,it was found that the expression of ATP6V0D2 in HCC samples was significantly higher than that in paracancerous controls and matched paracancerous controls.(2).Through survival analysis,it was found that the overall survival time and disease-specific survival time of patients with high expression of ATP6V0D2 HCC were significantly shorter than those of patients with low expression of ATP6V0D2,while the expression of ATP6V0D2 did not affect the progression-free survival of patients with HCC.(3).In the constituent ratio of clinical characteristics of patients with high and low ATP6V0D2 expression of HCC,the proportion of patients with histological grade G1+G2 and body weight > 70 kg was higher in the low expression group,while the proportion of histological grade G3+G4 and weight ≤ 70 kg was higher in the high expression group.(4).Through the correlation analysis between ATP6V0D2 expression and clinical characteristics,it was found that the higher the histological grade of patients,the higher the median expression of ATP6V0D2.In addition,the expression of ATP6V0D2 in patients with HCC ≤ 60 years old was significantly higher than that in patients over 60 years old.(5).Univariate and multivariate Cox regression analysis showed that ATP6V0D2 could be used as an independent indicator of poor prognosis in patients with HCC.(6).ROC curve analysis showed that with ATP6V0D2 expression 0.036 as the cutoff value,the sensitivity of predicting HCC was 90.6%,the specificity was 78.7%,the Yoden index was 0.787,and the area under the curve was 0.853.(7).Genome set enrichment analysis showed that the pathways that may be activated in patients with high expression of ATP6V0D2 are AKT signal pathway,cell cycle signal pathway,MAPK signal pathway,mTOR signal pathway and cancer-related signal pathway,while in patients with low expression of ATP6V0D2,the pathways that may be activated are drug metabolism pathway,fatty acid metabolism pathway and peroxisome proliferator activated receptor signal pathway.(8).The analysis of co-expressed genes by Linkedomics database showed that,the top 10 genes related to the positive expression of ATP6V0D2 were TP6V0D2,TM4SF19,C12ORF49,TREM2,GLI1,SULT1C2,NRIP3,MAPK13,STX3,and ADAM12,and all of them were high-risk prognostic factors of HCC.The top 10 negatively correlated genes were GRHPR,DCXR,CYB5D2,ADH1 B,AQP9,NUDT6,PEX11 G,ALDH2,ALAD,and MTHFD1,all of which were low risk prognostic factors for HCC.(9).The top 5 kinases with the highest co-expression correlation with ATP6V0D2 are protein kinase,Src family tyrosine kinase,cyclin-dependent kinase 1,checkpoint kinase 2,and serine/threonine protein kinase polo-like kinase,all of which are high-risk prognostic factors of HCC.Five Transcription factors,AP1,E2F1,E2F4,CP-2,and BACH2,are associated with ATP6V0D2 and have high risk in HCC survival prognosis.(10).Analysis of TIMER database showed that the expression level of ATP6V0D2 was negatively correlated with the Purity of immune infiltration,and positively correlated with the infiltration of B cells,CD4+T cells,macrophages,neutrophils and dendritic cells.Cox analysis showed that ATP6V0D2 had an independent prognostic effect on patients with HCC and was not affected by the immune infiltrating cells mentioned above.(11).The top five immunomarker genes positively correlated with ATP6V0D2 were MPZL1,CCT6 B,ADAM12,FABP5 and AQP9.The top five immunomarker genes with negative correlation were Clec5 a,Ca Pg,CCl14,St3Gal6 and Asgr1,respectively.2.Study on the effect of ATP6V0D2 on the Biological function of Hepatocellular carcinoma cells(1).The expression of ATP6V0D2 in HCC samples was significantly higher than that in the paracancer sample by RT-q PCR and IHC.(2).CCK-8 assay showed that the proliferation ability of hepatoma cells(Hep-3B and Huh-7)was significantly enhanced after overexpression of ATP6V0D2 gene,on the contrary,after knockout of ATP6V0D2 gene,the proliferation ability of hepatoma cells was significantly decreased.(3).Transwell chamber invasion and metastasis experiments showed that the invasion and migration ability of hepatocellular carcinoma cells were enhanced after overexpression of ATP6V0D2 gene,on the contrary,after knockout of ATP6V0D2 gene,the invasion and migration ability of hepatocellular carcinoma cells were significantly decreased.(4).Flow cytometry showed that the proportion of apoptosis of hepatoma cells decreased after overexpression of ATP6V0D2 gene,and increased significantly after knockout of ATP6V0D2 gene.(5).The results of ELISA showed that the ATPase activity of hepatoma cells increased after overexpression of ATP6V0D2 gene,on the contrary,the V-ATPase activity of hepatoma cells decreased after knockout of ATP6V0D2 gene.(6).The mRNA and protein expressions of LC-3B and Beclin-1 were decreased,while the mRNA and protein expressions of AKT and mTOR were increased in hepatoma cells overexpressing ATP6V0D2 and HCC samples with ATP6V0D2 positive.In contrast,the mRNA expressions of LC-3B and Beclin-1 were increased,while the mRNA expressions of Akt and mTOR were increased in hepatoma cells with ATP6V0D2 knockdown.ConclusionsThe study shows that the expression of ATP6V0D2 is significantly increased in HCC patients,which may serve as a potential diagnostic indicator of HCC and an independent high-risk indicator for the prognosis of HCC patients;the expression of ATP6V0D2 is also related to immune infiltrating cells and tumor infiltrating lymphocytes.Overexpression of ATP6V0D2 can promote the proliferation,migration,invasion and ATPase activity of hepatoma cells.In combination,ATP6V0D2 may play a role in the formation and development of HCC through activation of AKT/mTOR and inhibition of autophagy signaling pathway.