METTL3 facilitates immunosurveillance by inhibiting YTHDF2-mediated NLRC5 mRNA degradation in endometrial cancer

Abstract

Background: N6-methyladenosine (m6A) methylation is the most abundant chemical posttranscriptional modification of mRNA, and it is associated with the regulation of the immune response to tumors. However, the function of m6A modification in the immune response to endometrial cancer (EC) remains unknown. Our study investigated the immunological role of methyltransferase-like 3 (METTL3) in EC and the underlying molecular mechanism.

Methods: We investigated the correlation between the expression of METTL3 and CD8 by using an endometrial tissue microarray cohort. Next, we investigated the role and mechanism of METTL3 in the immune response to EC using a mouse tumor model and a CD8⁺ T cell-EC cell coculture system after METTL3 overexpression or depletion. Additionally, RNA immunoprecipitation (RIP), methylated RIP, and RNA stability experiments were used to investigate the mechanism underlying the function of METTL3 in immunosurveillance of EC.

Results: METTL3 levels were downregulated in EC patients, low levels of METTL3 were correlated with poor prognosis in EC patients. There was a positive correlation between METTL3 expression and CD8 expression. Overexpression of METTL3 in the EC cell and CD8⁺ T cell coculture system inhibited EC cell proliferation, migration, and promoted CD8⁺ T-cell proliferation, and in vivo, METTL3 overexpression increased CD8⁺ T cell proportions and inhibited EC progression; however, genetic depletion of METTL3 exerted the opposite effects. NLR family CARD domain-containing 5 (NLRC5) was identified as a target of METTL3-mediated m6A modification. The degradation of NLRC5 was increased by YTH domain-containing family 2 (YTHDF2).

Conclusions: Overall, METTL3, YTHDF2, and NLRC5 have potential to be the diagnostic and prognostic biomarkers for EC. METTL3 facilitated the m6A modifications of NLRC5 and inhibited its degradation through a YTHDF2-dependent mechanism in EC. Genetic overexpression of METTL3 attenuated the immune evasion of EC by promoting NLRC5-mediated immunosurveillance, suggesting that the METTL3/YTHDF2/NLRC5 axis is a promising target of immunotherapy in EC.

Keywords: Biomarker; Endometrial cancer; Immunosurveillance; Immunotherapy; N6-adenosine-methyltransferase-like 3; N6-methyladenosine; NLR family CARD domain-containing 5; YTH domain-containing family 2.

Fig. 1

METTL3 inhibits EC by promoting immunosurveillance. A The endometria from endometrial tissues microarray cohort was subjected to IHC analysis to determine METTL3 level (scale bar: 100 μm; **P < 0.01 vs. Normal group). B The protein and mRNA expressions of METTL3 in endometria from normal subjects and EC patients (n = 3, **P < 0.01 vs. Normal group). C METTL3 expression in EC form TCGA datasets (****P < 0.0001 vs. Normal group). D Kaplan-Meier curves of correlation of METTL3 with the EC cumulative survival from endometrial tissues microarray cohort (P = 0.04) and TCGA datasets (P < 0.001). E The endometria from endometrial tissues microarray cohort was subjected to IHC analysis to determine CD8 level (scale bar: 100 μm; **P < 0.01 vs. Normal group). F Expression correlation between METTL3 and CD8 in endometrial tissues microarray cohort by Pearson correlation analysis (R = 0.78, P < 2.2e-16). G The protein and mRNA expressions of METTL3 in HEC-1 A, HEC-1B, and Ishikawa cell. H The protein and mRNA expressions of METTL3 in Ishikawa cell with or without METTL3 overexpression (**P < 0.01 vs. Vector group). I The protein and mRNA expressions of METTL3 in HEC-1 A and HEC-1B cell with or without shMETTL3 (**P < 0.01 vs. shNC group). J The proliferation of CD8⁺ T cell in co-cultured system (**P < 0.01 vs. shNC group, ^##P < 0.01 vs. Vector group). K The proliferation of HEC-1 A, HEC-1B, and Ishikawa cell in co-cultured system (*P < 0.05 vs. shNC group, **P < 0.01 vs. shNC group, ^##P < 0.01 vs. Vector group). L The apoptosis of HEC-1 A, HEC-1B, and Ishikawa cell in co-cultured system (**P < 0.01 vs. shNC group, ^##P < 0.01 vs. Vector group). M The migration of HEC-1 A, HEC-1B, and Ishikawa cell in co-cultured system (scale bar: 100 μm; **P < 0.01 vs. shNC group, ^##P < 0.01 vs. Vector group). N Representative images of xenograft tumors in BALB/C female mice by Ishikawa cell transfected with or without METTL3 overexpression. O Average tumor volume of BALB/C female mice (**P < 0.01 vs. Vector group). P Average tumor weight of BALB/C female mice (**P < 0.01 vs.vector group). Q Frequency of CD8⁺ T cell in the peripheral blood of BALB/C female mice (**P < 0.01 vs. Vector group). R IHC staining of METTL3 and CD8 in solid tumor of BALB/C female mice (scale bar: 100 μm; **P < 0.01 vs. Vector group)

Fig. 2

METTL3 inhibits YTHDF2-mediated NLRC5 mRNA degradation. A m6Avar database of m6A modification on NLRC5 mRNA sequence and the very high confidence m6A site location (4808 and 5682) on NLRC5 mRNA sequence. B MeRIP-PCR data in HEC-1B cell shows the relative quantity of NLRC5 mRNA immunoprecipitated by the m6A antibody (m6A-IP) and IgG in cells with or without shMETTL3 (**P < 0.01 vs. shNC group), and the NLRC5 mRNA m6A modification site. C RIP-PCR data in Ishikawa cell with or without METTL3 overexpression shows the content of NLRC5 mRNA immunoprecipitated by METTL3 antibody (**P < 0.01 vs. IgG group). D NLRC5 expression in EC form endometrial tissues microarray cohort (scale bar: 100 μm; **P < 0.01 vs. Normal group) and TCGA datasets (*P < 0.05 vs. Normal group). E Kaplan-Meier curves of correlation of NLRC5 with the EC cumulative survival from TCGA datasets (P = 0.01). F Expression correlation between METTL3 and NLRC5 in endometrial tissues microarray cohort by Pearson correlation analysis (R = 0.63, P = 5.9e-12). G The protein and mRNA expressions of NLRC5 in HEC-1 A and HEC-1B cell with or without shMETTL3 (**P < 0.01 vs. shNC group). H The stability of NLRC5 mRNA in HEC-1 A and HEC-1B cell with or without shMETTL3 (**P < 0.01 vs. shNC group). I The protein and mRNA expressions of NLRC5 in Ishikawa cell with or without METTL3 overexpression (**P < 0.01 vs. Vector group). J The stability of NLRC5 mRNA in Ishikawa cell with or without METTL3 overexpression (**P < 0.01 vs. Vector group). K IHC staining of NLRC5 in solid tumor of BALB/C female mice (scale bar: 100 μm; **P < 0.01 vs. Vector group). L YTHDF2 expression in EC form endometrial tissues microarray cohort (scale bar: 100 μm; **P < 0.01 vs. Normal group) and TCGA datasets (*P < 0.05 vs. Normal group). M The protein and mRNA expressions of YTHDF2 in endometria from normal subjects and EC patients (n = 4, **P < 0.01 vs. Normal group). N Kaplan-Meier curves of correlation of YTHDF2 with the EC cumulative survival from TCGA datasets (P = 0.01). O Expression correlation between YTHDF2 and NLRC5 in endometrial tissues microarray cohort by Pearson correlation analysis (R = -0.59, P = 2.2e-10). P RIP-PCR data shows the content of NLRC5 mRNA immunoprecipitated by YTHDF2 antibody (**P < 0.01 vs. IgG group). Q The protein and mRNA expressions of YTHDF2 and NLRC5 in HEC-1B cell with or without YTHDF2 overexpression (**P < 0.01 vs. Vector group). R The stability of NLRC5 mRNA in HEC-1B cell with or without YTHDF2 overexpression (**P < 0.01 vs. Vector group). S The protein and mRNA expressions of YTHDF2 and NLRC5 in HEC-1B cell with or without shYTHDF2 (**P < 0.01 vs. shNC group). T The stability of NLRC5 mRNA in HEC-1B cell with or without shYTHDF2 (**P < 0.01 vs. shNC group). U The protein and mRNA expressions of NLRC5 in HEC-1B cell with or without METTL3 overexpression or METTL3 + YTHDF2 overexpression (**P < 0.01 vs. Vector group, ^##P < 0.01 vs. METTL3 overexpression group). V The stability of NLRC5 mRNA in HEC-1B cell with or without METTL3 overexpression or METTL3 + YTHDF2 overexpression