Construction Of Kinases Gene Co-Expression Network In Glioma And Regulation Of Tumor-Associated Macrophages By Identified Immune-Associated Kinase Gene HCK

Objective: Glioma is the most common malignant primary brain tumor in adults,among which Glioblastoma of WHO grade IV has the highest malignant degree,and the5-year relative survival rate of GBM is about 5%.The prognosis of the wild-type subtype of IDH gene is significantly lower than that of the IDH mutant type.hematopoietic cell kinase(HCK),a member of the SRC family of kinases,is mainly expressed in myeloid cells and B lymphocyte lineages.In a variety of tumors,HCK participates in a variety of signal transduction pathways to regulate the function of macrophages and other immune cells,contributing to the formation of immunosuppressive tumor microenvironment.This paper aims to elaborate the regulation and mechanism of HCK on the polarization,migration and infiltration of macrophages in tumor microenvironment based on the co-culture system of primary glioma cells.Methods:(1)The GBM cohort of the Cancer Genome Atlas(TCGA)and the transcriptome data of patient kinase genes from the Chinese Glioma Genome Atlas(CGGA)cohort were analyzed.Weighted gene co-expression network analysis(WGCNA)was used to identify the gene sets with high covariation in IDH wild type GBM and obtain the modules closely related to IDH wild type GBM.STRING database and Cytoscape are used to mine the interactions between genes and correlation analysis of immune microenvironment score screen for the acquisition of Hub gene HCK.Kaplan-Meier analyzed the relationship between HCK expression and prognosis in GBM patients.Gene ontology(GO)and gene set enrichment analysis(GSEA)were used for functional analysis.EPIC analysis,Estimate analysis,Xcell analysis and Timer analysis were used to evaluate the correlation between HCK and immune microenvironment.(2)The expression of HCK in different cell subsets of GBM patients in GEO database was analyzed.(3)Construction of co-culture system: Human mononuclear cell line THP-1 was cocultured with primary neurosphere GSC21 cells after being induced to become M0 by Phaborgite PMA,and M0 after co-culture was defined as TAM.The expression of STAT3 and p-STAT3 in macrophages of TAM and M0 in non-co-culture groups was detected by WB.q PCR and WB were used to identify the M1 or M2 phenotypes of macrophages.(4)HCK knockdown stable cell lines were constructed: lentiviruses of sh-HCK and NC were transfected with THP-1,and transfection efficiency was detected by q PCR and WB.THP-1 of NC and sh-HCK was induced into M0 by PMA and co-cultured with primary nerve bulb GSC21 cells.The M1 or M2 phenotypes of TAM were identified by q PCR and WB.(5)Transwell test was conducted to detect the effects of HCK on TAM migration and invasion ability.(6)The combination of HCK and STAT3 in TAM was detected by Co-IP experiment.Results:(1)A total of 15 network modules were obtained through WGCNA.MEmidnightblue module was highly correlated with IDH wild-type GBM,and HCK was found to be the hub gene of GBM IDH WT module by using STRING database.GBM patients with high expression of HCK had a poor prognosis.HCK was highly positively correlated with immune score and matrix score,and highly negatively correlated with tumor purity.GSEA analysis found that HCK regulates leukocyte migration,activates immune response and other pathways,and the downstream pathway of HCK includes JAK-STAT3.The macrophages with high HCK expression had higher x CELL fraction than those with low HCK expression.EPIC scores of macrophages with high HCK expression were higher than those with low HCK expression.(2)HCK was found to be highly correlated with M2-type macrophages in the GBM tumor microenvironment in the GSE84465 single-cell dataset.(3)M0 induced by THP-1 was co-cultured with GSC21.Compared with M0 without co-culture,it was found that M0 after co-culture was TAM and polarized towards M2-type macrophages,and the expression of HCK and p-STAT3 increased.(4)In the co-culture system with GSC21 induced by THP-1,M0 in sh-HCK group was polarized toward M1-type macrophages compared with the NC group.(5)In the co-culture system,TAM invasion and migration ability of sh-HCK group was significantly decreased compared with NC group.(6)Co-IP experiment detected the direct binding of HCK and STAT3.Conclusions: In glioma microenvironment,HCK expression in macrophages induces M2 polarization of macrophages through STAT3 pathway and promotes migration and invasion of macrophages.