Preliminary Study On The Mechanisms Of GPT2 Regulating The Biological Behavior Of Bladder Cancer

Objective(s): Glutamic-pyruvic transaminase 2(GPT2)catalyzes a reversible transamination reaction between alanine and α-ketoglutaric acid(α-KG)to produce pyruvate and glutamate during cellular glutamine catabolism and has been shown to play a role in different tumor occurrence and development.However,the regulatory role of GPT2 in bladder cancer is unclear.In this study,we explored the differential expression of GPT2 in bladder cancer and paraneoplastic tissues and the correlation with DNA methylation,gene mutation,tumor microenvironment,and immune cell infiltration by bioinformatics techniques.The effects of targeted inhibition of GPT2 gene expression on bladder cancer cell proliferation,migration,EMT,stemness,and subcutaneous tumorigenesis and development in nude mice were investigated by cellular and animal experiments.Methods: 1.We used different data from databases such as TCGA,CCLE,and GTEx to analyze the differential expression of GPT2 in BCa and paraneoplastic tissues and the correlation with gene mutation,DNA methylation,TME,and immune cell infiltration through a series of bioinformatics approaches to explore the potential role of GPT2 in BCa.2.The difference in the expression of GPT2 in BCa and paracancerous tissue was verified by immunohistochemical detection of GPT2 expression in BCa and paraneoplastic tissues.3.Western blotting was used to detect GPT2 protein expression in normal human bladder epithelial immortalized cells(SV-HUC-1)and human bladder cancer cell lines(5637,T24,J82)to verify the difference in GPT2 expression in the bladder epithelial cells and BCa cell lines.4. GPT2 knockdown and null lentiviral vectors were constructed and transfected with T24 bladder cancer cells to construct a stable transgenic strain.5.The effects of targeted inhibition of the GPT2 gene on proliferation,invasion,cycle,apoptosis,and stemness ability of BCa cells were examined by CCK-8 assay,clone formation,scratch assay,Transwell assay,flow Annexin V-FITC/PI,stem cell sphere-forming assay,CD44 antibody staining,and other cellular assays.6.RT-q PCR and WB assays were used to detect changes in m RNA and protein expression levels of genes related to epithelial-mesenchymal transition and stemness in bladder cancer cells after the knockdown of GPT2 gene expression levels.7.The subcutaneous tumor-bearing model of nude mice was established to explore the effects of targeted inhibition of GPT2 gene expression on the occurrence and development of subcutaneous transplantation tumors in nude mice.Results: 1.The GPT2 gene was significantly more highly expressed in BCa tissues compared to paraneoplastic or normal tissues and was associated with poor prognosis in BCa patients.2.The expression level of the GPT2 gene was significantly negatively correlated with DNA methylation,suggesting that the high expression of the GPT2 gene in bladder cancer may be due to its promoter region demethylation.3.Through GO and KEGG enrichment analysis of 300 genes positively associated with GPT2,we found that GPT2,in addition to being involved in amino acid metabolism,may be associated with tumorigenesis,gene expression regulation,immune cell infiltration,and cell proliferation.4.We analyzed the relationship between the GPT2 gene and the tumor microenvironment and showed that high GPT2 expression was significantly associated with features related to antigen processing,mismatch repair,nucleotide excision repair,DNA damage repair,DNA replication,base excision repair,epithelial-mesenchymal transition,and stemness maintenance.5.We analyzed the relationship between GPT2 expression and infiltration score and 26 types of infiltrating immune cells in bladder cancer through the Immu Cell AI database.The results showed that the expression level of GPT2 was negatively correlated with the infiltration score and negatively correlated with multiple infiltrates of most immune cells,except naive CD8+ T cells.6.Immunohistochemistry and Western Blot validation results show high expression of GPT2 in bladder cancer tissues and bladder cancer cells.7.GPT2 knockdown induces cell cycle arrest in bladder cancer cells to promote their apoptosis.8.Knockdown of the GPT2 gene inhibits epithelial-mesenchymal transition of bladder cells and reduces invasion and migration of bladder cancer cells.9.Decreased stemness of bladder cancer cells after knockdown of GPT2 gene.10.Knockdown of GPT2 gene expression in bladder cancer cells inhibits subcutaneous tumorigenesis and progression in nude mice.Conclusion(s): 1.The GPT2 gene is highly expressed in bladder cancer tissues and bladder cancer cells compared to paraneoplastic tissues and is associated with poor prognosis in patients with bladder cancer.2.GPT2 may be associated with bladder carcinogenesis,immune cell infiltration,cell proliferation,epithelial-mesenchymal transition,and stemness maintenance.3.Knockdown of the GPT2 gene down-regulated epithelial-mesenchymal transition and stemness of bladder cancer cells,inhibiting their proliferation,invasion,and migration as well as subcutaneous tumor formation and growth in nude mice.