Screening And Experimental Verification Of Ubiquitinated Genes Related To Immune Infiltration In Stage Ⅲ And Ⅳ Hepatocellular Carcinoma Based On Bioinformatics Analysis

Objective: Immune checkpoint therapy(ICIs)is one of the important modalities for the treatment of stage III and IV hepatocellular carcinoma(HCC),which can effectively improve the prognosis of HCC patients.However,its objective response rate(ORR)is less than 20%,which greatly limits the use of ICIs in patients with liver cancer.The level of tumor immune cell infiltration is an important factor affecting the response rate of ICIs.Recent studies have shown that ubiquitination genes play a key regulatory role in tumor immune infiltration.Therefore,studying the key molecules of ubiquitinated genes regulating immune infiltration of liver cancer can provide new targets for improving the efficacy of ICIs in HCC patients.This study aimed to explore the key ubiquitination genes that regulate immune infiltration in stage III and IV hepatocellular carcinoma,and to further experimentally verify them.Methods: The data were downloaded from The Cancer Genome Atlas(TCGA)and the patients were divided into three immune subgroups by single sample gene set enrichment analysis(ss GSEA),in which weighted gene correlation network analysis(WGCNA)identified modules of co-expressed genes correlated with immune infiltration.GO and KEGG analyzed the modules with the highest degree of immune infiltration.Then,based on TCGA,ICGC and STRING database,protein-protein interaction(PPI),stage analysis,Kaplan-Meier(K-M)curve were used to screen ubiquitinases associated with prognosis and HCC staging.In addition,ss GSEA,single gene sequencing and MCP counter were used to verify the relationship between GRB2 expression and prognosis and immune infiltration.We also explored the association of gene expression levels with gene mutations,drug treatment efficacy,and potential pathways.At last,immunohistochemistry,q RT-PCR,western blot were performed for vitro validation.Results: Based on the infiltration of immune cells,the TCGA patients were divided into three subgroups.In total,we identified 30ubiquitination-related proteases,after differential expression and WGCNA analysis.Subsequently,it was found that only the ubiquitinase GRB2,whose expression was not only proportional to prognosis,but also correlated with stage III and IV HCC.Then,the expression of GRB2 in patients with stage III and IV HCC was validated and found that patients with high GRB2 expression had deeper levels of worse prognosis,higher gene mutation rates and were more sensitive to immune checkpoint inhibitors.What’s more,GRB2 expression was positively correlated with the infiltration of immune cells(CD4 conv T cells,Treg cells,T prolif cells,CD8 +T cells and CD8 Tex cells)and was often enriched in immune-related pathways such as B cell proliferation.Finally,we verified that GRB2 was highly expressed at m RNA and protein levels in patients with stage III and IV by vitro experiments.Conclusions: The ubiquitinated gene GRB2 is significantly associated with the prognosis of patients with stage III and IV HCC.The higher the expression of GRB2,the higher the infiltration of tumor immune cells.New metrics for program evaluation.13 figures,2 tables,50 references...