The Mechanism Of RNA M~6A Methylation Modification In Patients With Recurrent Implantation Failure

Objective:To investigate the expression of RNA N6-methyladenosine(m~6A)modification-related enzymes in endometrial tissues of patients with recurrent implantation failure(RIF)during the"window"of implantation.To investigate the relationship between m~6A methylation and RIF.The transcriptomic m~6A modification profiles of RIF patients and their control endometrial tissues were obtained for the first time,and the differentially methylated genes and differentially expressed genes between the two groups were further analyzed.The key genes were screened to investigate their functional mechanisms in regulating the proliferation and senescence of human endometrial stromal cells(h ESCs).METHODS:The GEO dataset was selected according to the screening criteria,and the expression matrix was obtained after normalization of the dataset to detect the expression of RNA m~6A modification-related enzymes in RIF.A total of 15 patients with RIF and their controls attending the Center for Reproductive Medicine of the First Hospital of Lanzhou University from May 1,2021 to July 30,2022 were collected according to the nadir criteria for"window period"endometrial sampling,and the endometrial samples were collected by real-time fluorescence quantitative polymerase chain reaction(real-time fluorescence quantitative PCR).The expression of RNA m~6A modification-related enzymes in RIF was detected by real-time fluorescence quantitative PCR(RT-q PCR).The methylation profile and expression profile of m RNAs in RIF were analyzed using methylated RNA immunoprecipitation sequencing(MeRIP-seq)and transcriptomic RNA sequencing(RNA-seq)techniques,and m RNAs that underwent differential methylation and differential expression were further screened using diff Reps software and edge R software.Prediction of the potential biological functions of differentially methylated m RNAs using gene ontology(GO)functional analysis and Kyoto encyclopedia of genes and genomes(KEGG)signaling pathway analysis,respectively.N6-methyladenosine methylation levels and expression levels of CFTR,HAS2,and ITGAD were detected by MeRIP-q PCR and RT-q PCR.The functions of HAS2 in regulating the proliferation and senescence of h ESCs were investigated by CCK-8 assay,Ed U incorporation assay,and senescence-associated beta-galactosidase(SA-β-gal)assay.The proteins bound to HAS2 were investigated by pull down and liquid chromatography-tandem mass spectrometry(LC-MS/MS)experiments.RESULTS:In the GSE26787 dataset methyltransferase RBM15(p<0.01)and METTL14(p<0.05)were significantly elevated in RIF,demethylase ALKBH5 was significantly elevated in RIF(p<0.05),and methylated reading protein YTHDF2(p<0.05)and YTHDF3(p<0.01)were were significantly elevated.There was no statistical difference in the general data between the two groups of patients(p>0.05).As detected by RT-q PCR technique,the demethylase ALKBH5(p<0.05)and methylation reading protein YTHDF3(p<0.05)were significantly elevated in RIF,and the expression of methylation reading protein HNRNPA2B1 was significantly downregulated in RIF(p<0.05).740 m~6A significantly upregulated peaks and 238m~6A significantly downregulated peaks were found in the RIF group.Most of the m RNAs with m~6A modifications in RIF had only one m~6A peak.The differentially methylated genes were mostly enriched in glutamatergic synapses,c GMP-PKG signaling pathway,adenosine monophosphate-activated protein kinase(AMPK)signaling pathway,and endocrine factors regulating calcium reabsorption,etc.HAS2in RIF had its own gene expression HAS2 was screened as a key gene by down-regulating its own gene expression and m~6A modification level in RIF.By Ed U assay and CCK-8 proliferation assay,silencing of HAS2 was found to inhibit the proliferation of h ESCs compared to the control group.By SA-β-gal assay and senescence-related gene(p16)expression assay,silencing HAS2 was found to promote the senescence of h ESCs compared to the control.133 proteins were significantly different in capture values in pull down combined LC-MS/MS experiments.Conclusion:The demethylase ALKBH5 and the methylated reading protein YTHDF3 were significantly elevated in endometrial tissues of RIF patients implanted in the"window phase"in both GEO validation and RT-q PCR validation.There were significant differences in m RNA m~6A methylation modification profiles between endometrial tissues of RIF patients and controls.Silencing of HAS2 inhibited proliferation and promoted senescence of h ESCs.HAS2 binds to multiple ribosomal proteins.