Exploration Of The Expression And Function Of X-ray Repair Cross Complementing Gene Family In Hepatocellular Carcinoma

BackgroundCancer is a class of high mortality diseases in clinical practice and is the second leading cause of death in the United States after heart diseases.In China,cancer incidence and mortality rates are also high.DNA damage plays an important role in the development and progression of cancer,and the X-ray repair cross complementing(XRCC)genes play an important role in DNA damage repair.In humans,a total of six XRCC genes have been identified(XRCC1,XRCC2,XRCC3,XRCC4,XRCC5,XRCC6).Currently,most studies on XRCC genes in cancer are focused on gene polymorphisms,Their expression effects on the development,progression,and prognosis of cancers including hepatocellular carcinoma(HCC)and their own biological functions are not clear.ObjectiveThe aim of this study was to investigate differences in the expression of XRCC gene family in various cancers and to analyze the relationship between its expression and the prognosis of different cancer species.We screened cancer species of multi-gene of XRCC gene family effects on and explored the signaling pathways and biological functions of their main effects in this cancer species,constructed a prognostic model based on XRCC family genes,and initially investigated the effects of XRCC1 gene on the biological behavior of hepatocellular carcinoma.Methods1.Pan-cancer expression information and clinical data of XRCC family genes from the downloaded TCGA TARGET GTEx dataset in the UCSC database were analyzed for expression differences between normal and cancerous tissues using unpaired Wilcoxon Rank Sum and Signed Rank Tests using R software.Log-rank test was used to analyze the prognostic association of gene expression levels with cancer species.2.To explore the function of XRCC genes in HCC,we downloaded the hepatocellular carcinoma dataset from the official websites of two databases,TCGA and ICGC,and combined the expression information and clinical data of XRCC series genes in hepatocellular carcinoma,as well as the follow-up data.Cluster grouping of hepatocellular carcinoma patients was performed using consistency clustering analysis to explore the differential genes between the two groups.Enrichment analysis was performed to explore the differences in signaling pathways and biological functions among the different clustered subgroups based on differentially expressed genes.In addition,Cytoscape software was used to construct protein interaction network constructs and Hub gene screening.Finally,we compared the differences in somatic mutations and immune infiltration between the two clustering subgroups.3.To explore the prognostic value of XRCC genes in HCC,we used Lasso-Cox regression to screen XRCC genes associated with prognosis in hepatocellular carcinoma based on the expression and prognostic data of XRCC family genes in TCGA,and calculated the risk score.The clinical data and risk scores were combined to construct XRCC gene HCC-related prognostic models.4.To further validate the results of bioinformatics analysis,we collected 132 primary liver cancer surgical specimens and 20 normal liver tissues adjacent to cancer from Xiangya Hospital of Central South University from January 2019 to December 2020,and their related clinical data to verify the expression of XRCC gene in cancer development and progression using RT-q PCR and to explore its association with clinicopathological parameters and prognosis.5.In vitro experiments,RNAi technology were performed to inhibit XRCC1 gene expression.Western Blot(WB)was performed to investigate the differences in XRCC1 expression and the changes in E-cadherin and N-cadherin expression after XRCC1 gene inhibition in HCC cell lines and normal liver cells.Transwell was performed to investigate the effect of XRCC1 on the migration and invasion ability of HCC cell lines Hepg2 and Sun449.Clone tablet experiment and CCK8 were performed to investigate the effects of XRCC1 inhibition on the proliferation ability of Hepg2 and Sun449.In vivo experiments,XRCC1 gene expression was interfered with cholesterol-modified si RNA to investigate its effect on tumorigenesis of transplanted tumors in nude mice.Results1.XRCC genes were differentially expressed in multiple cancers and correlated with overall survival time and disease-free survival time in patients with multiple tumors.Among them,the expression of all six XRCC genes in hepatocellular carcinoma was increased in HCC,and their high expression suggested a poor prognosis in HCC.2.In HCC(both TCGA and ICGC databases),patients could be classified into two subcategories,high expression(C1)and low expression(C2),based on the XRCC gene expression level.subcategory C1 had a worse prognosis than subcategory C2.There were 271 differential genes between the two subclasses,and enrichment analysis showed significant differences in multiple metabolic pathways and biological functions between the two subclasses.the mutation rates of genes such as TP53,TTN,CTNNB1,MUC16,and ALB were higher in somatic cells of the C1 subclass than in the C2 subclass.Finally,the infiltration abundance of B cells,CD4+ T cells,neutrophils,macrophages,and myeloid dendritic cells was significantly higher in subclass C1 than in subclass C2.3.Univariate prognostic analysis and Lasso-Cox regression analysis,XRCC2,XRCC3,XRCC4,and XRCC5 were independent risk factors for liver cancer prognosis,and their high expression suggested poor prognosis.Nomogram was constructed to predict the prognosis of patients with hepatocellular carcinoma by combining patient age,gender,T-stage,clinical stage,pathological grading and risk score.4.The m RNA levels of XRCC1,XRCC2,XRCC3,XRCC4,XRCC5 and XRCC6 were higher in hepatocellular carcinoma than in normal liver tissue adjacent to cancer.However,only high expression of XRCC1,XRCC2,XRCC3,and XRCC6 was associated with more advanced clinical stage,worse pathological grading,and poor prognosis in patients with hepatocellular carcinoma in this batch of pathologies.5.The protein expression level of XRCC1 was significantly higher in HCC cell lines than in normal cells.Interfering with XRCC1 can inhibit the invasion,migration,epithelial mesenchymal differentiation phenotype in Hepg2 and Sun449 cells,and the growth of transplantation tumors in nude mouse of Sun499 lines.Conclusions1.XRCC genes are up-regulated in expression in multiple cancers and individual gene has potential as prognostic markers in multiple cancers.All XRCC family genes are upregulated in HCC expression and suggest a poor prognosis of HCC patients.2.Based on the XRCC genes transcript level,HCC can be divided into two clustered subgroups of high and low expression,which differ significantly in expression profile,biological function,signaling pathway,gene mutation landscape,and immune infiltration,and different expression group suggests a different prognosis of HCC patients.3.A prognostic model based on XRCC gene family has high promise in predicting the prognosis of HCC patients.4.The important role of XRCC gene family in HCC was further validated in a cohort of Chinese Hepatocellular Carcinoma patients.XRCC1,XRCC2,XRCC3 and XRCC6 m RNA levels were associated with HCC recurrence and could be a biological marker for predicting HCC prognosis.5.The results of both in vitro and in vivo experiments indicate that XRCC1 is an oncogene that promotes the proliferation,invasion and migration of HCC.