DataSheet_1_Expression of gasdermin D in clear cell renal cell carcinoma and its effect on its biological function.zip

BackgroundClear cell renal cell carcinoma (ccRCC) is the most common type of renal cell carcinoma, which suffers from the lack of diagnosis and treatment methods, and many patients cannot be diagnosed at first time. Gasdermin D (GSDMD) is involved in inflammatory reactions and pyroptosis and is considered a potential therapeutic target. This paper’s aim is to elucidate the expression of GSDMD in clear cell renal cell carcinoma and its value for treatment and prognosis, as well as its impact on the biological function of clear cell renal cell carcinoma.MethodThe Cancer Genome Atlas (TCGA) database was used to compare the expression of GSDMD in tumor and normal tissues, analyze its correlation with cancer stage and overall survival time, and establish receiver operating characteristic (ROC) curve, which was confirmed by the Gene Expression Omnibus (GEO) database and immunohistochemical staining of clinical samples and PCR and Western blotting (WB) of cell lines. The relationship between GSDMD and patient prognosis and staging was analyzed using TCGA database and validated using clinical sample data. Differentially expressed genes (DEGs) and epithelial–mesenchymal transition (EMT)-related genes of GSDMD were screened by TCGA database. Protein–protein interaction (PPI) of GSDMD was constructed by GeneMANIA and STRING, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were analyzed by the Metascape database. Then, R software was used to analyze the immune cell infiltration, immune microenvironment score, and tumor mutational burden (TMB) analysis of GSDMD high- and low-expression groups in TCGA database. GSDMD lentivirus was used to transfect 769-P cells to construct stable upregulated and downregulated transfected cell lines. PCR was used to verify the expression differences of differentially expressed genes between the high- and low-expression groups of GSDMD; then, MTT, flow apoptosis, and Transwell were used to detect the proliferation, apoptosis, invasion, and migration of the transfected cells.ResultsThe results of bioinformatics analysis showed that the expression of GSDMD in clear cell renal cell carcinoma was significantly correlated with patient stage and overall survival, and the tumor with high expression of GSDMD had a worse stage and overall survival. GSDMD has some significance in the diagnosis of ccRCC. The results of EMT correlation analysis and enrichment analysis showed that GSDMD was correlated with genes and pathways related to invasion and metastasis of renal cell carcinoma. The subsequent immune cell infiltration analysis showed that there were many differences in the infiltration of immune cells between the high- and low-expression groups of GSDMD, such as naive B cells. The immune microenvironment score showed that the high-expression group had a lower proportion of stromal cells than the local expression group but had a higher proportion of immune cells. Through TMB, it was shown that the high-expression group had a higher mutation. The expression of GSDMD in renal cell carcinoma by immunohistochemistry and in vitro cell experiments was confirmed. According to the prognostic information of clinical patients, it was found that GSDMD was significantly correlated with TNM stage, Fuhrman grade, lymph node metastasis, gender, and smoking or not, and the prognosis of patients with high expression of GSDMD was worse. After that, we constructed stable transfection cell lines with high expression and knockdown through lentivirus transfection and verified the expression amount of differentially expressed genes by PCR, which is consistent with the results of TCGA database. Then, we confirmed that GSDMD is related to proliferation, invasion, migration, and apoptosis of ccRCC by MTT, flow apoptosis, and Transwell assay. The low expression of GSDMD inhibits the proliferation, invasion, and migration of tumors and enhances apoptosis and vice versa. Therefore, GSDMD can be used as a potential biological marker for the diagnosis and prognosis of ccRCC.