Study On The Mechanism Of ERCC4 Gene Regulatory Network In The Immune Regulation Of Colorectal Cancer

Part 1:Based on Bioinformatics Analysis of Characteristic Genotype Mutations and Potential Immune Invasion and Prognostic Value in Patients with Colorectal cancerObjective:Immune cell infiltration plays a vital role in immune homeostasis.Various immune cell infiltrations can regulate tumor progression through complex tumor-immune interaction mechanisms.Understanding the status of tumor immune infiltration and discovering new immune-related prognostic biomarkers are issues that need to be resolved urgently.Materials and methods:We used next-generation sequencing(NGS)technology to detect the gene panle associated with tumor characteristics,and collected the clinical parameter data of the corresponding patients.Use bioinformatics technology to analyze the bio-enrichment function and correlation of the genes in the panle.Then we used the Tumor Immunity Assessment Resource Database(TIMER2.0)to analyze and compare the differences in expression,prognosis,immune infiltration and immune cell markers in CRC tumor tissues and adjacent normal colon tissues.Finally,we used the UALCAN database to analyze the promoter methylation levels of differential genes.Results:The mutant genes are enriched into three types:proliferation genedriven type,metabolic gene-driven type and microenvironmental gene-driven type.The comparison of the three types of genes before and after standardization shows that microenvironment driver genes have a greater weight in CRC.Analyze mutant genes and clinical information to obtain immune-related differential genes(ESR2,KDR,NRAS,ERCC4,CYP2C19,MSH2 and ROS1(P<0.05,P<0.05,P<0.05,P<0.05,P<0.05,P<0.05 and,P<0.05)).After analysis using the TIMER database,it was found that the expression of ERCC4 in colon adenocarcinoma(COAD)was higher,and it was positively correlated with the prognosis(P<0.01).Then we analyzed the relationship between ERCC4 and immune infiltration,the results showed that ERCC4 and B cells(R=0.342,P<0.001),CD8+T cells(R=0.336,P<0.001),CD4+T cells(R=0.444,p<0.001),macrophages(R=0.503,P<0.001),neutrophils(R=0.46,P<0.001)and dendritic cells(R=0.458,P<0.001)The degree of infiltration was positively correlated.To further explore the relationship between ERCC4 and immune cells,the analysis of immune cell markers showed that ERCC4 interacts with B cells,CD8+T cells,neutrophils,macrophages,dendritic cells,NK cells,Th1 cells,and T cells in COAD.The cell surface markers of Treg and monocytes are significantly positively correlated.Finally,the UALCAN database was used to analyze the ERCC4 promoter methylation level in COAD,and the results showed that the ERCC4 promoter methylation level in tumor tissues was significantly increased compared with that in normal tissues adjacent to cancer.Not only that,the methylation level of the ERCC4 promoter in the elderly and overweight people was higher than that in the control group(P<0.05).The methylation level of the ERCC4 promoter in men was higher than that in women(P<0.05).Conclusion:The expression of ERCC4 mRNA may be an important factor involved in the immune microenvironment and its enhancement of immune infiltration in COAD.Kaplan-Meier survival analysis results show that the ERCC4 gene has prognostic value for the OS of COAD,and the ERCC4 gene alone can be used as a prognostic molecular biomarker of COAD.Part 2:To Explore the Effect of Different Expression Levels of ERCC4 in Colon Adenocarcinoma Cells on MHC-I MoleculesObjective:This study is to analyze the possible effects of overexpression and silence of ERCC4 on immune cells in colon adenocarcinoma cells and its value as an immunotherapy sensitizer.Materials and methods:First,we carried out silencing and overexpression virus vector transfection on human colon adenocarcinoma cell line(DLD-1),and used RT-qPCR and Western blot to detect the expression level of ERCC4 gene.Then we again used RT-qPCR and Western blot to detect the expression level of MHC-I molecules on the transfected DLD-1 cells as one of the adaptive ligands for CD4+T cells and CD8+T cells,and analyzed ERCC4 Express the value of immunotherapy for CO AD.Results:First,the colon cancer cell DLD-1 was transfected with a lentiviral vector containing overexpression and silencing,and finally constructed into HDLD-1,NC1-DLD-1;L-DLD-1,NC2-DLD-1 Two groups of four colon cancer cell lines.The results of RT-qPCR showed that the expression of ERCC4 in NC2DLD-1 cells at the mRNA level was significantly higher than that in L-DLD-1 cells(P<0.05),and the expression in NC1-DLD-1 cells was significantly lower than that in H-DLD-1 cells(P<0.01).Western blot results showed that overexpression of ERCC4 at the protein level(P<0.01)and silence P<0.05)were equally successful.Then the mRNA and protein expression levels of HLAA,HLA-B and HLA-C of different cell lines were detected by RT-qPCR and Western blot.The results showed that the mRNA levels of HLA-A and HLA-C in NC1-DLD-1 increased by 2.44 times(P<0.05)and 1.54 times(P<0.05)compared with H-DLD-1.HLA-A,HLA-B and HLA-C mRNA levels in H-DLD1 cells were significantly higher than those in L-DLD-1 cells(P<0.05,P<0.05 and P<0.01).The protein expression of HLA-A in H-DLD-1 was 1.68 times higher than that of NC1-DLD-1(P<0.01).Compared with NC2-DLD-1 cells,the protein expression of HLA-C in L-DLD-1 was reduced by 48.63%(P<0.01).In addition,the protein expression levels of HLA-A,HLA-B and HLA-C in L-DLD1 were reduced by 26.89%(P<0.01),31.66%(P<0.01)and 37.23%respectively compared with H-DLD-1(P<0.05).Conclusion:In the cell experiment verification,it was found that HLA-A,HLA-B and HLA-C are positively correlated with changes in ERCC4 expression.It suggests that ERCC4 can increase the infiltration of immune cells by enhancing the recognition of MHC class I molecules.The combination of MHC-I molecules with CD4+T cells and CD8+T cells helps strengthen the immune system’s ability to recognize and present tumor antigens.ERCC4 may be an independent immunotherapy sensitization target for COAD,and ERCC4 has a certain prognostic value for colon adenocarcinoma,but further research is needed to confirm it in the future.