Exploring The Prognostic Value And Biological Function Of H2AFY Gene In Hepatocellular Carcinoma

Objective:To study the expression and prognostic value of H2AFY gene in hepatocellular carcinoma(HCC),explore its functional networks in HCC,and its effects on HCC cell proliferation,migration,cell cycle and apoptosis,and related signaling pathways.Methods:(1)TIMER,Oncomine and HCCDB online databases were used to analyze the expression differences of H2AFY in HCC tissues and normal liver tissues.The RNA-seq transcription profile data and corresponding clinical information of 371 and 232 HCC cases were downloaded from the TCGA and ICGC databases,respectively;the R language was used to extract H2AFY expression data and clinical information data,and analyze the distribution of H2AFY expression in clinical characteristics sub-groups.Logistic regression method was used to analyze the relationship between H2AFY expression and clinicopathological characteristics;K-M survival analysis,univariate and multivariate Cox regression analysis were used to investigate the relationship between H2AFY expression and prognosis.(2)The LinkedOmics online database was used to perform statistical analysis on H2AFY co-expression using the Spearman test in the TCGA HCC cohort,and GO term annotation,KEGG pathways,kinase-target enrichment,miRNA-target enrichment and transcription factor-target enrichment analyses were performed through Gene Set Enrichment Analysis(GSEA).(3)The expression level of H2AFY in normal hepatocytes and hepatocarcinoma cell lines was detected by RT-qPCR,and HepG2 and Hep3B cells were selected to construct the stable H2AFY knockdown cell lines by lentiviral transfection finally,the efficiency of knockdown was detected using Western Blot and RT-qPCR.CCK8 assay,clone formation assay,flow cytometry,cell scratch assay and Transwell cell migration assay were applied to investigate the effect of H2AFY knockdown on cell proliferation,apoptosis,cell cycle and migration of HCC cells.Finally,the expression of related signal pathway proteins was detected by Western Blot.Results:(1)The results in multiple databases indicate that the expression of H2AFY in HCC tissues is significantly higher than that in normal liver tissues,and is significantly correlated with age,survival status,histopathological grade,tumor size and TNM stage.The overall survival(OS)of patients with high expression of H2AFY is significantly shorter than low expression patients,and H2AFY can act as an independent prognostic indicator for HCC.(2)The H2AFY co-expression networks include 7201 significantly positively correlated and 2928 significantly negatively correlated genes(FDR<0.01).Notably,the top 50 significantly positive genes showed the high likelihood of being high-risk genes in HCC,in which 34/50 genes were with high hazard ratio(HR)(HR>1,p<0.05).In contrast,there were 11/50 genes with low HR(HR<1,p<0.05)in the top 50 negatively significant genes.GSEA showed that H2AFY co-expressed genes involved mainly in microtubule cytoskeleton organization in mitosis,organelle fission,cell cycle,homologous recombination,and spliceosome and other biological processes.(3)The expression of H2AFY in HCC cells is up-regulated compared with normal liver cells.H2AFY knockdown can inhibit the activation of the STAT3 pathway,significantly reduce the proliferation ability of HCC cells,increase the proportion of apoptosis,weaken the migration ability,and arrested cell cycle in G0/G1 phase.Conclusions:(1)The expression of H2AFY in HCC tissues is significantly higher than normal liver tissues,and high H2AFY expression associates with poor prognosis in HCC.(2)H2AFY and its co-expressed genes are mainly involved in regulation of cell cycle,homologous recombination,and mitosis and other key biological processes.(3)Knockdown of H2AFY can inhibit the activation of STAT3,thereby inhibiting the proliferation and migration of HCC cells,promoting apoptosis and arresting cell cycle in G0/G1 phase.