Regulation Of PD-L1 Through RNA M~6A Modification In Bladder Cancer Cells

Bladder Cancer(BCa)is the fourth most common urinary Cancer.In addition to surgery,recurrent or advanced bladder cancer mainly needs to be treated with chemotherapy.However,due to its high recurrence rate,the treatment cycle is longer,and the huge medical expenses bring great pain to the patients.In recent years,drugs related to tumor immune checkpoint inhibitors PD-1 and PD-L1 have been approved for the clinical treatment of bladder cancer.However,PD-1/PD-L1 blocking monotherapy is only effective in about 20%of patients with bladder cancer.PD-L1 is a target of tumor immunotherapy,and its expression level is related to the efficacy of immunotherapy to a certain extent.Therefore,further understanding of the regulatory mechanism of PD-L1 will help improve the efficacy of immune checkpoints,thus benefiting patients with bladder cancer.Currently,there are few reports on epigenetic regulation of PD-L1 expression,especially at the RNA modification level.Our previous study found that the expression of RNA m~6A modifying enzyme METTL3 was abnormally elevated in bladder cancer,and it was found that METTL3 promoted the occurrence and development of bladder cancer by regulating the mRNA m~6A levels of MYC,Rela,IKB?B and AFF4.In addition,we also found that m~6A signal was significantly enriched near the termination codon of PD-L1 mRNA in bladder cancer cells,and the change of METTL3 expression caused the corresponding change of PD-L1 level.Therefore,we inferred that the expression of PD-L1 was also regulated by the methylation of mRNA m~6A.In this paper,MERIP-q PCR test showed that m~6A modification was enriched at specific sites of PD-L1 3’UTR,and the m~6A modification level of PD-L1 was reduced after METTL3 knockdown,and the luciferase reporter gene activity containing PD-L1 3UTR region was also regulated by METTL3.Thus,it was revealed that METTL3 promoted the expression of PD-L1 by regulating the modification level of its RNA m~6A.In addition,knockdown of the RNA m~6A reading protein IGF2BP1 significantly reduced PD-L1 mRNA and protein levels in both types of bladder cancer cells,suggesting that IGF2BP1 mediated RNA degradation regulates PD-L1expression in BCA.Lactate dehydrogenase(LDH)assay and apoptosis assay demonstrated that downregulation of METTL3 in bladder cancer cells can enhance the killing sensitivity of BCA cells to T cells by decreasing the expression of PD-L1.In vivo experiments,we proved through BBN-induced BCA model that regulating the expression of METTL3 can effectively reduce the expression of PD-L1 and enhance the immune response of bladder cancer in vivo.Therefore,our data reveal the expression mechanism of PD-L1 in tumor cells and provide ideas for improving the efficacy of immunotherapy.