| Objectives:By combining single cell RNA sequencing and differential gene expression analysis,analyze the impact of EGFR gene mutation on the immune microenvironment of lung cancer,in order to provide new ideas for clinical treatment of patients with EGFR mutation.Methods:Combined with the TCGA database to analyze the impact of EGFR expression on the prognosis of patients.The chest-occupying patients who were newly treated were screened according to the inclusion and exclusion criteria.After signing informed consent,tissue samples were obtained by surgery or puncture.Then took histochemical detection to confirm the mutation of EGFR gene.At the same time,completed the preparation and quality control of the single-cell suspension of the sequencing sample,continued to perform single-cell separation,library preparation and sequencing on the samples that met the quality control standards,obtained the corresponding single-cell transcriptome information,and used R language and other tools to express the data matrix,secondary quality control,gene annotation,cell clustering,differential gene enrichment and functional enrichment,etc.to complete data analysis and display in visual charts.Results:1.The survival analysis of lung adenocarcinoma data in the TCGA database found that the median survival time of patients with high expression of EGFR was not significantly different from that of low expression group(p>0.05);2.Among the 12 newly diagnosed chest mass-occupying patients who met the inclusion criteria,after excluding 3 non-adenocarcinoma samples and 3 substandard quality control samples,6 samples were used for subsequent sequencing analysis,and2 of them were negative for EGFR gene mutation,4 cases were positive for EGFR mutation;the cell maps of 6 samples were constructed respectively,with a total of56439 cells and 30 cell clusters.3.8 cell types were obtained by marker gene clustering,namely epithelial cells,endothelial cells,fibroblasts,proliferating cells,B cells,plasma cells,T cells,and myeloid cells;among them,the EGFR mutation-negative group mainly clustered In the EGFR mutation-positive group,the proportion of epithelial cells was lower,but the proportion of myeloid cells,plasma cells and T cells was significantly increased.4.Cell subtype clustering was performed on myeloid cells and T cells,and 8myeloid cell subtypes and 5 T cell subtypes were obtained respectively.In the EGFR mutation-positive group,p DC cells(plasmacytoid dendritic cells)and CD4~+naive T(CD4~+naive T cells)were higher than those in the mutation-negative group,while the ratio of CD4~+Treg(CD4~+regulatory T cells)was lower than that in the mutation-negative group.5.The differential gene expression analysis of the EGFR mutation-positive group and the negative group first-level cells was screened to obtain the top ten differentially expressed genes in each first-level cell.Further analysis of secondary cells found that IGHG4 was upregulated in T cells,myeloid cells,and fibroblasts in the EGFR mutation-positive group.6.GO functional enrichment and KEGG analysis of the differential genes between the EGFR mutation-positive and negative groups showed that the down-regulated genes in myeloid cells were mainly enriched in the positive regulation of the immune effector process and the processing and presentation of MHC-Ib antigens.It is suggested that EGFR mutation may lead to the inhibition of myeloid cell immune activation.Conclusions:1.EGFR mutation leads to an increase in the proportion of myeloid cells,plasma cells and T cells in lung adenocarcinoma;among them,the proportion of myeloid cell subtype p DC cells and T cell subtype CD4~+naive T increases,while the proportion of CD4~+Treg subtype decreases.2.The expression of IGHG4 was up-regulated in T cells,myeloid cells and fibroblasts of EGFR mutation-positive group;3.The EGFR mutation-positive group may mediate the immunosuppression of the tumor microenvironment by inhibiting the positive regulation of myeloid cells and the processing and presentation of MHC-Ib antigens. |
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