Study On MRNA M6A Methylation Modification And Expression Profile In Villi Tissue Of Patients With Early Embryonic Arrest

BACKGROUNDEmbryo arrest in early pregnancy is a common pathological pregnancy,which belongs to a special form of Spontaneous abortion(SA).The common clinical view is that SA is caused by a series of subclinical diseases(anatomical abnormalities,endocrine changes,thrombosis,latent infection and immune disorders,etc.),and is in a "fragile" early pregnancy state,thus miscarriage.In addition,there are still about half of Recurrent spontaneous abortion(RSA)whose causes have not been ascertained.The lack of relevant information on the causes undoubtedly limits the guidance and treatment before pregnancy.Epigenetic changes have been shown to play a key role in embryonic development,placental function and reproductive system diseases.In recent years,as one of the most popular RNA modifications in the field of epigenetics research,m6 A methylation has not only exploded in tumor research,but also suggested that it may be related to germ cell and embryo development.Among them,the overall expression profile of m6 A modified mRNA in RSA villus tissue has not been reported yet.OBJECTIVEThe aim of this study was to systematically analyze the changes of m6 A methylation pattern and mRNA expression profile in the early,unexplained embryonic arrested villus tissue,and to construct the mRNA expression map of m6 A modification in the unexplained embryonic arrested villus tissue.Combined analysis of mRNA expression and m6 A methylation was conducted to explore the role of m6 A methylation in the pathogenesis of early unexplained embryonic arrest.METHODS(1)Recruiting patients(RSA group)and normal early pregnancy patients who have voluntarily terminated their pregnancy(NC group)in XX Hospital from June2019 to August 2020 due to multiple embryo abortions for unexplained reasons.Collect 30 g of their miscarried villi groups,and store them at-80℃ after quick freezing in liquid nitrogen.(2)Total RNA was extracted by Trizol method in both groups,and stored in-80℃ refrigerator after qualified quality inspection.The RNA was divided into two parts,one part to construct the transcriptome sequencing library,and the other part to construct the Me RIP-seq library after enrichment of m6 A specific antibody.Qualified library sequencing.Quality control and preliminary analysis of sequencing data were carried out.(3)After Me RIP-seq,the differential peaks are screened,and the genes related to the differential peaks are analyzed by GO and KEGG functional enrichment.The data obtained after RNA-seq was normalized and analyzed for differences,and significantly different genes were screened for volcano map analysis and clustering heat map analysis,and functional enrichment analysis for DO,GO,and KEGG was performed.All of the above use FDR<0.05 and |log2FC|>1 as the screening conditions.(4)Combined analysis of Me RIP-seq and RNA-seq data.Genes with significant changes in m6 A modification level and mRNA expression level were screened out,and GO enrichment and KO enrichment analysis were performed for the differentially expressed genes.(5)RT-q PCR was performed on the m6 A regulators METTL3,HNRNPC and the6 selected genes whose m6 A modification level and mRNA expression level changed significantly at the same time.(6)Statistical analysis was performed by SPSS22.0 software and Graph Pad Prism 7.0 software.Measurement data were measured as mean±standard error(M±SD).The comparison between the RSA group and the NC group was performed by t test.P<0.05 considered the difference to be statistical Learn meaning.RESULTS(1)The m6 A peaks of the RSA group and the NC group are mainly distributed in the CDS and 3’UTR areas,and the most common motif in the m6 A peak area is GGACU.(2)Among the 4382 peaks identified by Me RIP-seq,according to the screening criteria of P<0.05 and |log2FC|>1,a total of 138 peaks with significant differences were screened,among which the m6 A modification of the RSA group was up-regulated compared to the NC group There are 101 peaks,and 37 peaks are reduced by m6 A modification.KO analysis of differential peak-related genes showed that the significant enrichment pathways mainly include Toll-like receptor signaling pathway,natural killer cell-mediated cytotoxicity,NOD-like receptor signaling pathway,herpes simplex virus infection,and apoptosis.(3)Among the total 20286 mRNA-related genes detected by RNA-seq,according to the P<0.05 and |log2FC|>1 screening criteria,a total of 3574 significantly different genes were screened,including 2720 significantly up-regulated genes and 854 significantly down-regulated genes.gene.The DO items related to the female reproductive system that are mainly enriched by these significantly different genes include embryonic development,choriocarcinoma,preeclampsia,infertility,etc.;pathways that are significantly enriched in KO analysis include Th17 cell differentiation,Ras signaling pathways,Jak-STAT signaling pathway,cancer-related pathways,anti-folate,apoptosis,etc.(4)Combined analysis of Me RIP-seq and RNAseq,with P<0.05 and |log2FC|>1as the screening conditions,a total of 65 genes with significant differences in m6 A modification and significant changes in mRNA levels were screened.(5)The expressions of m6 A methylation regulators "writers" METTL3 and "readers" HNRNPC were significantly down-regulated(P<0.01),and the expressions of other regulators did not change significantly.(6)RT-q PCR verification,except for the EMILIN1 verification results,which showed no significant difference,the other genes METTL3,HNRNPC,MTR,LGI2,ZBTB4,TFAP4 and PLSCR3 were all consistent with the mRNA sequencing results(P<0.05).CONCLUSIONS(1)The motif bound by m6 A methylation regulator in human villi and the functional areas modified by m6 A were consistent with those of other species and were conserved.(2)m6A methylation is differentially modified in early embryonic arrest villi.(3)Abnormal m6 A modification leads to significant changes in at least part of mRNA expression levels,which may lead to reduced proliferation,insufficient implantation and increased apoptosis of the hairy tissue in patients with RSA,thus causing the arrest and death of early embryos.(4)Our study delineated the m6 A methylation modification map of the early embryonic villus tissue in arrest of development,and provided a new perspective for exploring the mechanism of embryonic arrest in patients with RSA.