Role And Mechanism Of JNK Signaling In Regulating The M~6A Methylation In Bladder Cancer

Background:Bladder cancer is the most common malignancy of the urinary system,easy to relapse,and has a poor prognosis.Bladder cancer causes nearly170000 deaths worldwide every year.In the past 40 years,the systemic treatment of muscle invasive and advanced bladder cancer has mainly been platinum-based chemotherapy and surgical treatment.In the past decade,immunotherapy has become a new and effective strategy for cancer treatment,particularly in the PD1/PD-L1 and CTLA-4 immune checkpoint inhibitors of tumor immunotherapy-based cancer treatment.However,although the FDA has approved the clinical treatment of bladder cancer sample a variety of immune checkpoint inhibitors,which only achieve efficacy in some patients,the majority of poor results in the remaining patients.Thus,there is need to further explore the regulatory mechanism of immune checkpoints in order to provide new ideas for tumor immunotherapy.Mitogen activated protein kinase(mitogen-activated protein kinase,MAPK)is a group of highly conserved in eukaryotes serine/threonine protein kinase,can be activated by different extracellular stimuli,such as cytokines,cytokines,Neurotransmitters,hormones,cell stress and cell adhesion activation.There are three major subfamilies of mitogen-activated protein kinases(MAPK):the extracellular-signal regulated kinases(ERK MAPK),the c-JUN N-terminal kinase or stress-activated protein kinases(JNK or SAPK),and MAPK14;among them,JNK is one of the important branches of the MAPK family.The JNK signaling pathway plays an important role in a variety of physiological and pathological processes such as cell cycle,reproduction,apoptosis and cell stress.However,more evidence shows that JNK has two-sidedness and regulates cell apoptosis and survival by cooperating with NF-κB,JAK/STAT and other signaling molecules.m~6A methylation modification is a prevalent m RNA species,each internally modified,mainly by METTL3,METTL14,WTAP like complex composed methylase and demethylase FTO,ALKBH5.More and more evidences show that the abnormal and genetic changes of m~6A methylation modification are related to the disorder of multiple biological processes such as abnormal cell death and proliferation,developmental defects,malignant tumor progression,impaired self-renewal ability and abnormal immune regulation.Epigenetic modification represented by m~6A methylation has important significance in tumor immune regulation.Previous studies in this laboratory have shown that up-regulated m~6A methylation can promote the occurrence and development of bladder cancer,but its upstream regulatory mechanism has not been clearly reported.This project explores the mechanism of the JNK signaling pathway regulating the level of m~6A methylation modification and its relationship with tumor immunity through in vitro and in vivo experiments,so as to provide new ideas for the clinical treatment of tumors.Objective:In this study,we constructed a chemically induced mouse model of bladder cancer and a mouse model of subcutaneous tumor formation to explore the relationship between JNK signaling pathway and m~6A methylation in vitro and in vivo,clarified that the JNK signaling pathway regulates the overall RNA m~6A methylation mechanism of bladder cancer cells by up-regulating the transcription of METTL3,and explored new strategies for the treatment of bladder cancer by inhibiting JNK signal activity.Method:First,we used the TCGA bladder cancer database for bioinformatics analysis to evaluate the correlation between JNK and METTL3.Then,the expression of METTL3 and PD-L1 in the bladder cancer cell lines 5637 and J82that were treated with JNK si RNA and SP600125,were detected by western blot and RT-qPCR.We used SRAMP software to predict the m~6A modification site of PD-L1 3’UTR online,and then used Me RIP experiment and m~6A kit to further detect the PD-L1 m~6A modification level and overall m~6A of 5637 and J82 cells treated with SP600125 and JNK-si RNA.We predicted the potential JNK downstream transcription factors that regulated the transcription of METTL3 online by PROMO software,and verified by Ch IP experiments.In addition,we also conducted cell function experiments.The 5637 and J82 cells treated with SP600125 and si RNA were co-cultured with T cells.The results showed that the T cell-mediated toxicity and apoptosis of bladder cancer cells were improved after the JNK signal reduced.In order to further confirm that JNK affects the expression of PD-L1 by regulating METTL3,we also conducted a Rescue experiment of overexpression of METTL3.In addition,we also used BBN chemically to induced orthotopic bladder cancer animal models and subcutaneous tumor ectopic bladder cancer animal models in C57BL/6 mice.Mouse were randomly divided into groups and received SP600125 treatment.We recorded body weight of mouse and tumor size every three days for analysis,and bladder samples were collected for immunohistochemical analysis.Result:In this study,we found that JNK signal was positively correlated with the expression of METTL3.For bladder cancer cell lines,JNK affects the level of m~6A methylation modification by regulating the expression of METTL3,and it is found that the down-regulation of JNK can increase the killing of bladder cancer cells by T cells.For BBN-induced mouse models and subcutaneous tumor models,tumor weight and volume growth were inhibited after SP600125treatment,and immunohistochemical analysis of bladder cancer samples treated with SP600125 also increased the expression of the apoptotic factor Ac caspase.The results of the above-mentioned in vitro experiments are verified.Conclusion:In this study,the upstream regulatory signal of m~6A methylation modification and the role of JNK inhibitors in the treatment of bladder cancer were found in vivo and in vitro for the first time.Down-regulation of JNK signal can inhibit the immune escape ability in bladder cancer.Our findings suggest a new treatment for bladder cancer.