Activation Of CGAS-STING Signal To Inhibit The Proliferation Of Bladder Cancer: The Immune Effect Of Cisplatin

Background Cisplatin is the most commonly used component of adjuvant chemotherapy for advanced bladder cancer.It was believed that the killing effect of cisplatin on tumor cells including bladder cancer mainly depended on its irreversible DNA damage.Recent studies have found that cisplatin can not only kill tumor cells directly through DNA damage,but also affect the immune components in tumor microenvironment through indirect immune effects,thereby affecting the proliferation levels of tumor cells,but its specific mechanism and role of these immune effects are still unclear.Thus,to explore the immune effect of cisplatin in bladder cancer is helpful to understand the comprehensive mechanism of cisplatin in bladder cancer patients.Objectives1.To explore the correlation between cisplatin related immune effects and the activation of endogenous c GAS-STING signal in bladder cancer and its regulatory mechanism2.To investigate the effect of cisplatin induced endogenous c GAS-STING signal on the proliferation of bladder cancerMethods1.In this study,we first found that cisplatin treatment significantly activated immune and inflammation related pathways in T24 bladder cell line through RNA transcriptome sequencing and bioinformatics analysis.Further analysis revealed that c GAS-STING signal may be closely related to the activation of these pathways.Objective to explore the basic expression level of STING protein in bladder cancer.real time q PCR,western blot,ELISA,reporter gene assay,immunofluorescence and flow cytometry analysis were used to verify the activation and downstream signals of c GAS-STING pathway in cisplatin treated T24 and TCCSUP cell lines.To verify whether the cisplatin related immune effects of T24 and TCCSUP cell lines were significantly reduced by knocking down STING or using STING specific inhibitor.2.To explore the basic expression level of the key upstream protein c GAS of STING in bladder cancer.Objective to explore the DNA damage and repair status of bladder cancer cell lines after cisplatin treatment by immunofluorescence,subcellular component western blot,and to explore whether the total expression level,the distribution of c GAS and the level of chromatin bound c GAS protein in bladder cancer cell lines after cisplatin treatment are changed.3.CCK-8 assay and clone formation assay were used to explore whether the proliferation levels of bladder cancer cell lines were changed after knocking down STING protein.Combined with the public database,we analyzed whether the expression levels of c GAS-STING key proteins in TCGA were related to the overall survival and disease-free survival of bladder cacner patients.4.Bladder cancer cell lines MB49 stably transfected with STING-knockdown and STING-WT were constructed by lentiviral transfection.The subcutaneous tumor model of MB49 bladder cancer was established in C57BL/6J mice.The proliferations of subcutaneous tumor in PBS+STING-knockdown group,PBS+STING-WT group,cisplatin + STING-knockdown group and cisplatin + STING-WT group were compared.The infiltration ratios of CD3 + CD45 +,CD8 + CD45 +,CD11 c + MHCII + and F4 /80 + CD11 b + cells were analyzed by flow cytometry.Combined with the above results,we analyzed the effect of cisplatin related STING signal activation on the proliferation of bladder cancer in mice and its possible mechanism.Finally,CIBERSORT was used to analyze the correlation between the prognosis of bladder cancer patients receiving cisplatin based chemotherapy and the level of immune cell infiltration in TCGA database.Results1.We found that cisplatin induced the immune and inflammation effects in bladder cancer cell line T24,which was mainly reflected in the enrichment of immune pathways such as type I interferon secretion and the significantly increased secretion of cytokines including IL-6 and INF-β.This special immune effect is closely related to the activation of c GAS-STING pathway.Cisplatin induced accumulation of ds DNA and release of chromatin bound c GAS protein in T24 and TCCSUP bladder cancer cell lines were the important reasons for activating c GAS-STING signal and leading to downstream immune effects.2.We found that knocking down STING protein did not affect the proliferation of T24 and TCCSUP bladder cancer cell lines in vitro.In the subcutaneous tumor transplantation experiment of C57BL/6J mice with healthy immune function,cisplatin activated endogenous c GAS-STING signal in bladder cancer cells inhibited the proliferation of subcutaneous tumor of bladder cancer in mice.The activation of endogenous c GAS-STING signal in bladder cancer cells induced the increase of infiltrating DCs and CD8 + T cells in subcutaneous tumor of bladder cancer in mice.Conclusion Cisplatin can activate endogenous c GAS-STING signal by inducing DNA damage and promoting the release of chromatin bound c GAS protein.Cisplatin induced activation of endogenous c GAS-STING signal in bladder cancer promoted the secretion of cytokines including IL-6 and INF-β,and significantly increased the number of infiltrating DCs and CD8 + T cells in subcutaneous tumor of bladder cancer in mice.This study will provide a new idea for cisplatin combined with immunotherapy in the treatment of advanced bladder cancer.