Study On The Mechanism Of Methyltransferase-like 3 In Regulating Immunosurveillance Microenvironment Of Endometrial Cancer

Background and objectiveEndometrial cancer(EC)is the most common cancer of the female reproductive system.The incidence of EC patients is increasing year by year,and the age of onset is younger.Traditional chemoradiotherapy has a poor therapeutic effect on high-grade EC patients,as well as recurrent and metastatic EC patients.Immunotherapy has shown a good effect in the treatment of EC patients,and has been widely carried out in clinical practice.However,there are still a large proportion of EC patients who are not sensitive to immunotherapy.Therefore,exploring the molecular biological characteristics of EC patients who are not sensitive to immunotherapy and finding the target which can regulate these molecular biological characteristics will contribute to improve the effect of immunotherapy in EC patients.N6-methyladenosine(m6A)modification is one of the most common mechanisms of chemical modification on RNA.m6A modification forms a reversible regulatory mode through its methyltransferase and demethyltransferase,and affects RNA translation,splicing,degradation and so on under the action of m6A binding protein.Studies have shown that m6A modification is intimately associated with tumorigenesis and development.Methyltransferase-like 3(METTL3)is the first identified methyltransferase of m6A modification.Previous studies have indicated that low-expressed METTL3 in EC patients can promote EC development by activating the proliferation of EC cell.Nevertheless,whether METTL3can regulate immune-related characteristics in EC patients remains unclear,as well as its regulatory mechanism.Our study aims to explore the immune surveillance effect and the underlying molecular mechanism of METTL3 in EC,further evaluate its predictive value in EC patients,and provide new ideas for clinical immunotherapy of EC patients.MethodsThe expression of METTL3 was detected by immunohistochemistry(IHC),Western blotting,and real-time quantitative polymerase chain reaction(RT-q PCR).Overexpressed or interfered with METTL3 expression in EC cell line by lentivirus transfection technique,CD8~+T cell was extracted by magnetic cell separation,and CD8~+T cell co-cultured with EC to form the immunomicroenvironment.CD8~+T cell proliferation was detected by carboxycein diacetate succinimidyl ester(CFSE)in the co-cultured system,cell counting kit-8(CCK-8)assay was used to detect the proliferation of EC cell in the co-cultured system,flow cytometry was used to detect the apoptosis of EC cell in the co-cultured system,and transwell and wound healing assay were used to detect the invasion and metastasis of EC cell in the co-cultured system.The changes of immune surveillance and tumorigenicity in BALB/C female mice were analyzed by tumorigenicity assay.Methylated RNA immunoprecipitation quantitative PCR(Me RIP-q PCR),RNA immunoprecipitation-q PCR(RIP-q PCR),and actinomycin assay were used to analyze the mechanism underlying METTL3 regulated immune surveillance in EC.ResultsMETTL3 was down-regulated in EC patients,and low expression of METTL3 was associated with poor prognosis in EC patients.The expression of METTL3 was positively correlated with CD8 expression.In the co-culture system of EC cell and CD8~+T cell,overexpression of METTL3 can inhibit the proliferation and invasion of EC cell,and promoting the proliferation of CD8~+T cell,while interference with METTL3 expression would lead to the opposite effect.Mechanistically,NLR family CARD domain containing 5(NLRC5)was confirmed to be the m6A modification target by METTL3.Moreover,METTL3-mediated modification of NLRC5 m RNA m6A was depended on the m6A binding protein YT521-B homology domain-containing family 2(YTHDF2).Finally,we found that overexpression of METTL3 or NLRC5 could promote immunosurveillance and inhibit the formation of solid tumors in BALB/C female mice.ConclusionsMETTL3 could facilitate m6A modifications of NLRC5 and inhibit its degradation via YTHDF2-dependent mechanisms in EC.The activation of NLRC5-mediated immunosurveillance could inhibit the occurrence and development of EC,signifying the METTL3/YTHDF2/NLRC5 axis is a potential target for immunotherapy of EC.