Immune Related Prognostic Indicator Based On TCGA Analysis And Its Antitumor Function In Lung Adenocarcinoma

Objective The incidence rate of lung adenocarcinoma(LUAD)is increasing rapidly.Traditional treatments include surgical resection,radiofrequency ablation,transcatheter arterial chemoembolization and chemoradiotherapy,which have limitations in improving patient survival.In this study,the prognosis related gene C-C chemokine receptor 2(CCR2)was postulated,after the immune microenvironment of LUAD was analyzed through the data of the Cancer Genome Atlas(TCGA).The antitumor effect of CCR2 was explored in CD8+tumor-infiltrating lymphocytes(TILs)in LUAD.These results provide novel direction and theoretical support for complementary treatment in LUAD.Methods Part I: Bioinformatics analysis of human LUAD data from TCGA database using R Language.1.The matrix components and immune components in the immune microenvironment of 551 cases of LUAD were scored by R-package "ESTIMATE",and the differentially expressed genes(DEGs)shared by matrix components and immune components were screened.2.Conduct gene ontology(GO)analysis and Kyoto Encyclopedia of genes and genomes(KEGG)enrichment analysis on DEGs to see the overall function of DEGs.3.Construct protein-protein interaction(PPI)network for DEGs and conduct univariate Cox analysis to screen the survival related DEGs and construct lasso regression model for further prognostic analysis.4.Gene set enrichment analysis(GSEA)was performed on the final target gene CCR2 to find the enriched pathway of the gene,and survival analysis and clinical correlation analysis were carried out.5.The ratio of immune cells in the immune microenvironment of each LUAD sample was calculated by R package "CIBERSORT",and the relationship between CCR2 and 22tumor-infiltrating immune cells(TICs)was analyzed by difference analysis.Part II:Verification of the expression and antitumor function of CCR2 at the cellular and animal levels.1.HE staining was used to detect histomorphology and infiltration of inflammatory cells in microinvasive adenocarcinoma(MIA),invasive adenocarcinoma(IAC)and mouse Lewis lung carcinoma(LLC)tissue.2.Immunohistochemistry was used to detect the expression of CCR2 and CD8 in MIA,IAC and LLC tissue.3.The co-expression of CD8 and CCR2 in MIA,IAC and mouse LLC tissue was detected by immunofluorescence.4.Construct LLC mouse model,isolate and purify CD8+T cells in peripheral blood,spleen and tumor tissue,and the expression of CD69,CD107 a and CCR2 molecules were detected by flow cytometry.5.CCR2 over-expression lentivirus was transduced with CD8+TILs.The expressions of CD69,CD107 a and CCR2 of LV-Con CD8+TILs and LV-CCR2 CD8+TILs were detected by flow cytometry.6.The migration ability was assessed by transwell experiment in LV-Con CD8+TILs and LV-CCR2 CD8+TILs.7.The apoptosis of LLC was detected by flow cytometry after LLC cells were co cultured with LV-Con CD8+TILs and LV-CCR2 CD8+TILs for 24 hours.8.The proliferation ability of LLC was detected by CCK-8 after LLC was co cultured with LV-Con CD8+TILs or LV-CCR2 CD8+TILs for 48 hours.Results CCR2 was selected as an immune related prognostic indicator of LUAD through bioinformatics analysis.GSEA results showed that the CCR2 high-expression group of C2-kegg gene set was mainly enriched in T cell receptor signaling pathway.The difference analysis in TICs showed that CD8+TILs were closely correlated to the expression of CCR2.CCR2 was differentially expressed between patients with LUAD and para-carcinoma tissue(P=0.011);the immunohistochemistry results of human LUAD showed that the expression of CCR2 was significantly negatively correlated with the grades of LUAD(P<0.001);The immunohistochemistry results of LLC mouse model showed that the expression of CCR2 was significantly negatively correlated with the grades of LUAD(P<0.001).CCR2 over-expression lentivirus was successfully transduced in CD8+TILs.The results of migration experiment showed that,the migration rate of LV-CCR2 CD8+TILs was higher than LV-Con CD8+TILs.The results of cell apoptosis showed higher early apoptosis rate in LV-CCR2 CD8+TILs group of LLC tumor cells,in comparison to LV-Con CD8+TILs group.The result of cell proliferation,showed higher inhibiting of the proliferation of LLC tumor cells in LV-CCR2 CD8+TILs group of LLC tumor cells,compared to LV-Con CD8+TILs group.Conclusion 1.As a core gene related to prognosis in the immune microenvironment of lung adenocarcinoma,CCR2 is an important prognostic immune indicator.It decreases with the increase of clinical pathological stage of luad,and is correlated with a variety of tics;2.CCR2 up regulation stimulates the early activation of cd8+tils,promotes their migration to the simulated luad immune microenvironment in vitro,promotes the early apoptosis of tumor cells,and inhibits the proliferation of tumor cells,so as to achieve some anti-tumor effect.