The Biological Study Of The TCIRG1 Gene In Renal Clear Cell Carcinoma

ObjectiveAmong the malignant tumors of the urinary system,clear cell renal cell carcinoma(ccRCC)lacks reliable biomarkers,and some patients with ccRCC have reached an advanced stage or metastasized at the time of initial diagnosis,leading to poor prognosis.CD8+T cells are one of the important components of the tumor immune microenvironment.The infiltration level of CD8+T cells is closely related to the prognosis of patients with ccRCC and the sensitivity of immunotherapy.Therefore,the purpose of this study is to screen a biomarker closely related to CD8+T cell infiltration in ccRCC based on the weighted gene co-expression network analysis(WGCNA)and a variety of bioinformatics analyses.MethodsThe transcriptome sequencing data and corresponding clinical information of 539 ccRCC tissue samples and 72 normal kidney tissue samples were downloaded from the Cancer Genome Atlas(TCGA)database.Download the microarray sequencing data and clinical information of corresponding tissues from the GSE53757,GSE66272,GSE40435,GSE167093,and GSE29069 datasets in the Gene Expression Omnibus(GEO)database.Based on the above sequencing data,TCIRG1,the hub gene closely related to CD8+T cell infiltration in ccRCC,was screened by using the "Cell-type Identification by Estimating Relative Subsets of RNA Transcripts(CIBERSORT)" immune cell infiltration algorithm and WGCNA.The differential expression of TCIRG1 was verified by western blotting of 7 ccRCC tissues and normal kidney tissues.Kaplan-Meier survival analysis,univariate and multivariate Cox regression analysis evaluated the prognostic value of the TCIRG1 gene in ccRCC.Pearson correlation analysis evaluated the correlation between TCIRG1 expression level and multiple immune cells and common immune checkpoint-related genes.Gene Set Enrichment Analysis from multiple data sets speculated the possible signal pathway of TCIRG1 gene enrichment and verified the analysis results through the western blotting assay.ccRCC cells 786-O and Caki-1 with stable knockdown of TCIRG1 gene expression were constructed by lentivirus interference vector transfection technology.The changes in the proliferation and migration ability of ccRCC cells after knockdown of TCIRG1 gene expression were evaluated by cell scratch assay,MTT assay,Transwell migration assay,cell cloning assay,flow cytometry assay,tumorigenesis assay and other in vitro and in vivo functional experiments.ResultsBy analyzing the results of WGCNA in the TCGA dataset and GSE53757 dataset,we screened 169 genes closely related to CD8+T cell infiltration in ccRCC,and then determined TCIRG1 as the hub gene based on protein interaction network,univariate and multivariate Cox regression analysis.The results of the differential expressed analysis showed that the expression level of the TCIRG1 gene in the transcriptome of ccRCC tissue samples was higher than that in normal kidney tissue,and the expression level of the TCIRG1 gene was significantly positively correlated with the malignancy of ccRCC.The western blotting assay of ccRCC tissue and normal kidney tissue confirmed that the protein expression level of the TCIRG1 gene in ccRCC tissue increased.The results of RT-q PCR also verified that the m RNA expression level of TCIRG1 in ccRCC cells(786-O and Caki-1)was higher than that in normal renal tubular epithelial cells HK-2.The Kaplan-Meier survival analysis of ccRCC patients in the TCGA dataset and GSE29069 showed that the higher the expression level of the TCIRG1 gene,the worse the prognosis of ccRCC patients.Univariate and multivariate cox regression analysis further confirmed the independent prognostic value of TCIRG1 in ccRCC.The results of the immunoassay showed that the increased expression level of the TCIRG1 gene could activate the immune system of ccRCC to a certain extent.Through a series of functional experiments in vitro and in vivo,it was proved that down-regulating the expression of the TCIRG1 gene can inhibit the proliferation and migration of 786-O and Caki-1 cells,and can inhibit the tumorigenicity of nude mice in vivo.Finally,Gene Set Enrichment Analysis and western blotting assay explained that the TCIRG1 gene may participate in the occurrence and development of ccRCC by regulating JAK/STAT signal pathway.ConclusionsTCIRG1 gene may play a carcinogenic role in ccRCC by regulating the JAK/STAT signal pathway.The increase of TCIRG1 gene expression level is closely related to the malignant progression and poor prognosis of ccRCC patients.TCIRG1 can not only become a potential biomarker for the diagnosis and prognosis of ccRCC but also provide a new choice for the target of gene therapy of ccRCC.