Expression Of HAPLN3 Gene In Clear Cell Renal Cell Carcinoma (ccRcc),its Prognostic Significance And Immune Characteristics:An Analysis Based On Bioinformatic Database

Objective:Based on multiple public databases such as The Cancer Genome Atlas(TCGA),Genotype-Tissue Expression(GTEx),and Immune Infiltration Analysis(TIMER),To analyze the correlation between hyaluronic acid and proteoglycan-binding linker protein gene family member 3(HAPLN3)in clear cell renal cell carcinoma(ccRCC).Methods:First,a pan-cancer analysis of the m RNA expression level of HAPLN3 was performed through the TCGA and GTEx databases.The TCGA database was used to obtain ccRCC m RNA data and corresponding clinical information,and R software was used to analyze the differential expression of HAPLN3 between WHO/ISUP class III(G3 group)and IV(G4 group).To explore the effect of HAPLN3 on ccRCC survival and the difference in survival between G3 and G4 subgroups.Univariate and multivariate Cox regression analyses were used to assess the relationship between HAPLN3 and five major factors,including age,p T stage,p N stage,p TNM stage,WHO class,and overall survival.The correlation between HAPLN3 and infiltration levels of various immune cell subtypes was analyzed based on the TIMER database.The association of HAPLN3 with various immune-related genes was assessed by gene co-expression analysis.Differences in immunotherapy between G3 and G4 subgroups were compared by immune checkpoint analysis and tumor immune dysfunction and exclusion(TIDE)score.Finally,GSEA enrichment analysis was applied to investigate the potential immune pathway of HAPLN3 in ccRCC.Human renal clear cell carcinoma cell lines OS-RC2,ACHN and human renal cortical proximal tubule epithelial cell line HK-2 were constructed,and the differential expression of HAPLN3 protein in renal cancer cells and normal cells was observed by WB.Results:Pan-cancer analysis found that HAPLN3 is indeed differentially expressed in breast cancer,prostate cancer,skin melanoma and other tumor tissues and their normal tissues,which is consistent with previous literature reports.It was found that the expression of HAPLN3 was also different in ccRCC and normal tissues,and the expression of HAPLN3 gene in clear cell renal cell carcinoma was significantly higher than that in normal renal tissue(P<0.001).In ccRCC with different WHO grades,the expression of HAPLN3 m RNA in G4 group was significantly higher than that in G3 group(P<0.01)and normal kidney group(P<0.001).Univariate and multivariate Cox regression analyses were used to construct prognostic models to assess HAPLN3 and major clinical factors.Finally,it was found that high expression of HAPLN3,advanced age,high p TNM stage and high grade were the factors of poor prognosis of patients.Further analysis of immune correlation revealed that the expression of HAPLN3 in ccRCC was closely related to immune score,stromal score,and tumor microenvironment by the XCELL algorithm(P<0.001).It was found that the highly expressed HAPLN3 gene was positively correlated with the infiltration of dendritic cells,neutrophils,B cells,CD8 T+ cells,macrophages and CD4+ T cells by the TIMER algorithm(P<0.05).Subsequently,in the further evaluation of the correlation of immune cell subtype infiltration,we found that HAPLN3 expression was correlated with multiple T cell subtypes,and was positively correlated with immune cell subtypes such as activated NK cells and macrophage M1(P<0.001).In the correlation analysis of immune checkpoints of different subtypes of ccRCC,it was found that CTLA4,LAG3,PDCD1,and TIGIT were more expressed in the G4 group.And HAPLN3 was also positively correlated with CTLA4,LAG3,PDCD1,TIGIT in gene co-expression analysis(P<0.01).GSEA enrichment analysis showed that HAPLN3 was significantly enriched in multiple immune-related pathways,including cytokine receptor interaction,NK cell-mediated cytotoxicity,chemokine signaling pathway,autoimmune thyroid disease,JAK_STAT signaling pathway,adhesion Molecular cam,hematopoietic cell line,T cell receptor signaling pathway,primary immune deficiency,B cell receptor signaling pathway,MAPK signaling pathway,TOLL-like receptor signaling pathway,Notch signaling pathway,cancer-related pathway,wnt signaling pathway,etc.The WB test of renal cancer cell lines showed that the expression level of HAPLN3 in OS-RC2 and ACNH renal cancer cell lines was significantly higher than that in HK-2 normal kidney cell line.Conclusions:Based on the bioinformatics database,HAPLN3 is up-regulated in ccRCC and co rrelated with histological grade,which may be an independent prognostic marker for c c RCC patients and has potential value as an immunotherapy target.