Expression Of M6A Methyltransferase METTL3 In Human Placenta And Its Role In Early-onset Severe Pre-eclampsia

Background N6-methyladenosine(m6A)is the most abundant methylation modification in eukaryotic m RNAs.It is a dynamic and reversible modification involving methyl trans ferase,demethylase and binding protein.Recently,it has been found that m6 A modification has a certain correlation with pre-eclampsia.However,there are few literatures and no relevant molecular mechanism report.Objects To confirm the correlation between m6 A modification and early onset severe preeclampsia(EO-s PE).In normal and EO-s PE placentas,the important proteins involved in m6 A modification were screened for their expression levels.To study the effect of this protein on the biological behavior of trophoblast cells.Explore the downstream molecule,and investigate its effect on the function of the upstream protein.Methods(1)Colorimetry was used to detect the level of m6 A quantitatively in normal and EO-s PE placentas.(2)Immunohistochemistry,qRT-PCR and Western-blotting were used to detect the expression level of METTL3 in normal and EO-s PE placentas.At the same time,the correlation between METTL3 m RNA level and m6 A level was analyzed.Small-interfering RNAs and lentiviral vector were used to down-regulated and up-regulated the expression level of METTL3 in trophoblast cells lines(HTR-8/SVneo and JEG-3 cell),and the m6 A level was quantified.CCK-8 assay,plate clone formation assay,wound healing assay and Transwell assay were used to study the effects of METTL3 on the biological behavior of trophoblast cell proliferation,migration and invasion.(3)Transcriptome sequencing,MERIP-q PCR,qRT-PCR and Western-blotting were used to screen the downstream genes whose m6 A modification and protein expression were regulated by METTL3.The dual luciferase reporter gene technique was used to verify the m6 A binding sites of METTL3 and its downstream gene.Actinomycin D chase assay was utilized to evaluated the m RNA half-life of the downstream gene.(4)Small-interfering RNAs was used to down-regulate the expression level of ERO1 A in HTR-8/SVneo cells.CCK-8 assay,plate clone formation assay,wound healing assay and Transwell assay were utilized to explore whether ERO1 A regulates the effects of METTL3 on the biological behavior of trophoblast cell proliferation,migration and invasion.Moreover,the expression level of ERO1 A in normal and EO-s PE placentas was preliminarily detected by immunohistochemistry and qRT-PCR.Results(1)The m6 A level and the expression level of METTL3 in EO-s PE placentas were both higher than normal placentas.In addition,there was a positive correlation between them.(2)In HTR-8/SVneo and JEG-3,down-regulating the expression level of METTL3,the m6 A level was reduced,and the ability of cell proliferation,migration and invasion was enhanced.On the contrary,up-regulating the expression level of METTL3,the m6 A level increased,and the ability of cell proliferation,migration and invasion was weakened.(3)After screening and verification,it was confirmed that ERO1 A was the possible downstream gene of METTL3.Down-regulating the expression of METTL3,the m6 A level of ERO1 A decreased,the m RNA half-life of ERO1 A increased,the m RNA and protein level of ERO1 A increased.Down-regulating the expression of ERO1 A,the ability of cell migration and invasion was weakened.When the expression of METTL3 and ERO1 A were down-regulated simultaneously,the enhancement degree of cell migration and invasion was decreased.(4)The expression level of ERO1 A in the placenta of EO-s PE was decreased.Conclusions The m6 A methyltransferase METTL3,may affect the biological behavior of trophoblast cells by negatively regulating the downstream gene ERO1 A,contributing to the pathogenesis of early onset severe preeclampsia.The possible mechanism is that the increased expression of METTL3 leads to the increased level of ERO1 A m6A,which leads to a decrease in the stability of ERO1 A m RNA and in the level of ERO1 A m RNA and protein,leading to a decrease in the migration and invasion ability of trophoblast cells,resulting in abnormal placental development,and promoting the occurrence of early onset severe preeclampsia.