Function Of ELK3 And Its Regulatory Mechanism In Glioma

Background: Glioma is the most common primary malignant tumor of the central nervous system,with high morbidity and mortality.Despite great advances in surgery,radiotherapy,and chemotherapy over the past few decades,the prognosis for glioma patients remains poor.To detect the underlying mechanisms of carcinogenesis and development of glioma and explore the therapeutic targets are the heat-spot for research.ELK3 is a transcription factor that belongs to the ETS transcription factor family.Members of the ETS family regulate many important biological processes,such as immune response,in normal and tumor cells.Studies have shown that ELK3 is associated with malignant progression and tumor-infiltrating immune cells in various tumors,leading to poor prognosis.However,its function and regulatory mechanism in glioma is unclear and needs further study.Objective:To explore the function of ELK3 and its regulatory mechanism in glioma.Methods:(1)We obtained glioma datasets from TCGA,CGGA and GEO databases and used differential analysis and WGCNA analysis to screen high-expressed genes in glioma.We used Kaplan-Meier survival curve,univariate and multivariate Cox regression to analyze the relationship between ELK3 expression and the prognosis of patients;(2)The expression of ELK3 in normal brain tissues and glioma tissues was detected by RT-q PCR and immunohistochemistry(IHC);(3)The effects of ELK3 on the proliferation,migration and invasion of glioma cells were detected by CCK-8 proliferation assay,clone formation assay,scratch assay,and Transwell invasion assay;(4)GO and KEGG functional enrichment analyses were used to explore the biological function of ELK3,and GSVA was used to estimate the relationship between ELK3 expression and tumor-infiltrating immune cells in the glioma microenvironment;(5)The relationship between the expression of ELK3 and macrophage marker CD163 in glioma samples was detected by IHC;(6)Correlation analysis was used to explore the relationship between IDH1 mutation status and Elk3 expression in glioma;IDH1 with different mutations was overexpressed in glioma cells to explore its effect on ELK3 expression.Results:(1)Analysis of TCGA,CGGA and GSE16011 datasets showed that ELK3 was highly expressed in glioma,and its expression level was positively correlated with the grade of glioma;Kaplan-Meier survival analysis showed that patients with high expression of ELK3 had a worse prognosis than those with low expression of ELK3;univariate Cox regression analysis showed that the high expression of ELK3 was significantly correlated with the poor prognosis of glioma patients(TCGA: 95%CI=2.75-5.11,P<0.001;CGGA: 95%CI=1.63-2.46,P<0.001);multivariate Cox regression analysis showed that ELK3 was a significant independent prognostic factor in glioma patients(TCGA:95%CI =1.08-2.28,P< 0.001;CGGA: 95%CI=1.14-1.77,P<0.001);(2)The results of RT-q PCR and IHC of clinical samples showed that ELK3 was highly expressed in glioma,and its expression level was positively correlated with the grade of glioma;(3)CCK-8 proliferation assay,clone formation assay,scratch assay and Transwell invasion assay showed that the proliferation,migration and invasion abilities of glioma cells were decreased after knocking down the expression of ELK3 in glioma cells;(4)Enrichment analysis indicated that ELK3 may regulate immune components in the glioma microenvironment;ESTIMATE algorithm results showed that glioma tissues with high expression of ELK3 had higher stromal scores and immune scores than glioma tissues with low expression of ELK3,indicating that the tumor purity of glioma tissues with high expression of ELK3 was lower;online TIMER results showed that the expression of ELK3 was positively correlated with the abundance of neutrophils and macrophages,and negatively correlated with tumor purity in low-grade glioma samples;GSVA results showed that the abundance of macrophages and neutrophils was positively correlated with the expression of ELK3;(5)In clinical samples,IHC results showed that the expression of ELK3 was positively correlated with the expression of macrophage marker CD163;(6)The expression of ELK3 in glioma was negatively correlated with the methylation level of ELK3;the methylation level of ELK3 in glioma tissues with wild-type IDH1 was lower than that in glioma tissues with mutant type IDH1,and the m RNA expression of ELK3 in glioma tissues with wild-type IDH1 was higher than that in glioma tissues with mutant type IDH1;RT-q PCR and Western blotting detection showed that the m RNA and protein expression levels of ELK3 were increased or decreased when wild-type IDH1 or mutant type IDH1 was respectively overexpressed in glioma cells(SHG44 and HS683)or in glioma cells(T98G and U251);RT-q PCR and IHC results showed that the expression level of ELK3 in the IDH1 wild-type group was higher than that in the IDH1 mutant group in clinical samples.Conclusions:(1)ELK3 is highly expressed in glioma and can be used as an independent prognostic factor;(2)The proliferation,migration and invasion of glioma cells are inhibited when the expression of ELK3 in glioma cells was knocked down;(3)The high expression of ELK3 may be associated with increased infiltration of macrophage in glioma;(4)In glioma,wild-type IDH1 is a potential driver of high ELK3 expression.