The LIN28B/let-7 Axis Regulates Preeclampsia By Controlling WTAP-Dependent N6-Methyladenosine Methylation Of FZD2 MRNA

Objective:Pre-eclampsia(PE)is a multi-systemic placenta-related disease during pregnancy that severely affects maternal and infant health and is one of the major causes of increased maternal and perinatal morbidity and mortality.Impaired trophoblastic cell invasion and remodeling of spiral arteries was considered as the specific pathogenesis of pre-eclampsia.Among the 170 different RNA transcriptome-level chemical modifications identified,N6-methyladenosine(m~6A)is the most common post-transcriptional modification of eukaryotic mRNAs and long-noncoding RNAs.N6-methyladenosine modification is characterized by highly conserved sequences near the m~6A modification motif,and m~6A modifications on mRNAs mainly occur in DRACH motif(where D to A,G or U;R to G or A;H to A,C or U).The dynamic modification of this particular motif is reversibly regulated by methyltransferase(Writer)and demethylase(Eraser).m~6A-modified RNA modified by m~6A can be recognized by methyl binding protein(Reader).m~6A modification has many functions in cells,and it is now known to be involved in stem cell differentiation,growth and development,DNA damage repair,heat shock response,tumorigenesis,immune response and other life activities.The regulatory enzymes of RNA m~6A modification in placentas of patients with PE,the RNAs affected by m~6A modification dysregulation,and the possible regulatory pathways involved can provide new clues for studies related to the regulatory mechanisms of RNA transcriptome in pre-eclampsia.To elucidate the potential m~6A modification mechanisms of mRNAs involved in the phenotypes and cellular signaling pathways of PE.In this study,we examined the m~6A modification levels of total RNA in placental tissue,m~6A modifying enzymes(METTL3,METTL14,WTAP,FTO,ALKBH5)and changes in trophoblast cell function,by studying early-onset preeclampsia,late-onset preeclampsia,and control placentas at corresponding gestational weeks.At the same time,RNA m~6A modifications in trophoblast cells after WTAP knockdown were mapped by Methylated RNA immunoprecipitation(MeRIP)and high-throughput sequencing,and candidate genes with significant m6A modifications were screened.WTAP expression was altered in vitro by knockdown and overexpression,then to detect the alteration of proliferation,invasion and migration ability of trophoblast cell.To verify the involvement of WTAP in the regulation of m~6A modification of FZD2 mRNA in sequencing candidate genes.The correlation of this study will provide important experimental evidence to further elucidate the involvement of RNA methylation m~6A modification in the pathophysiological mechanisms of early-onset preeclampsia.Methods:(1)Detection of m~6A modification levels and m~6A modifying enzymes in early-onset preeclampsia,late-onset preeclampsia and control placenta at corresponding gestational weeks:(1)Collecting tissue specimens of early-onset preeclampsia,late-onset preeclampsia and control placenta at corresponding gestational weeks;(2)Using colorimetric method,quantitative real-time PCR and Western Blot to detect m~6A modification levels,methyltransferases and demethylases(METTL3,,WTAP,METTL14,ALKHB5 and FTO)in total RNA of early-onset preeclampsia,late-onset preeclampsia and placental tissues of control gestational weeks,and compare with public database bio-information using bioinformatics to screen key methylation modification-related genes associated with pre-eclampsia.(3)Expression of m~6A modifying related enzyme WTAP in cells after LIN28B/let-7a-2-3p overexpression and knockdown.(2)Association analysis of m~6A epigenomic information profile and RNA sequencing with MeRIP sequencing after WTAP knockdown in HTR-8/SVneo:(1)WTAP knockdown and control groups were selected for MeRIP high-throughput sequencing to detect mRNA genes with significant m~6A modification differences(Fold change>1.5,P<0.05)and their enrichment(m~6A methylation level and m~6A quantity);(2)using RNA high-throughput sequencing technology to screen genes that were significantly differentially expressed in WTAP knockdown and control groups for enrichment and pathway analysis;(3)To screen for candidate genes by performing a combined analysis of RNA sequencing and MeRIP sequencing to reveal m~6A modification-regulated mRNAs(m~6A methylation level and m~6A quantity isotropic changes,with altered RNA sequencing gene expression levels).(3)the effect of WTAP-regulated m~6A modification changes on the altered phenotype of proliferation,infiltration,and migration ability of trophoblast cell lines HTR-8/SVneo and SWAN-71 cells:(1)constructing small interfering si-RNA and overexpression lentiviral vector,transfected HTR-8/SVneo and SWAN-71 cells,respectively,and detected the altered phenotypes of proliferation,invasion and migration ability of HTR-8/SVneo and SWAN-71 cells and downstream pathway target genes by Ed U,flow cytometry,q RT-PCR and WB techniques;(2)Constructing knockdown FZD2 si-RNA,transfecting HTR-8/SVneo,SWAN-71 cells,and detect the alteration of proliferation,invasion and migration ability of HTR-8/SVneo and SWAN-71 cells and the expression of downstream pathway target genes using Ed U,flow cytometry,WB,etc.;(4)Statistical analysis:the expression of HTR-8/SVneo,SWAN-71 cells were detected using SPSS 26.0(SPSS,Inc.,Chicago,USA)and Graph Pad Prism 9.0 software(Graph Pad,Inc.,La Jolla,CA,USA)for statistical analysis.Experiments were repeated at least three times independently.The measured data were expressed as mean and/or SD.The t-test was used for the analysis of data on continuous variables with two independent normal distributions;the nonparametric Mann-Whitney test was used for data that did not conform to a normal distribution;also,the nonparametricχ2 test was used to analyze data on categorical variables.P values for each analysis are marked on the numbers,and the level of statistical significance was defined as P<0.05(*P<0.05;**P<0.01;***P<0.001).Results:(1)Alterations in m~6A modification levels and related enzyme expression in early-onset preeclampsia,late-onset preeclampsia and placental tissues at corresponding gestational weeks.(1)The total RNA m~6A methylation modification level of placental tissues in early-onset preeclampsia was lower than that in the control group;no significant difference was seen between late onset preeclampsia and late onset control.(2)The downregulation of WTAP may be the reason why the m~6A modification level of total RNA was decreasing;(3)Compared with the control group,the total RNA m~6A methylation modification level in the early onset control group was higher than that in the late onset control group suggesting that RNA m~6A may be in the placenta development with temporal fluctuations,and WTAP downregulation is involved in the regulation of m~6A methylation modification levels of total RNA in placental tissues of this disease.We selected WTAP knockdown HTR-8/SVneo cell lines for MeRIP sequencing and RNA sequencing.(2)Combined with the LIN28B protein,which was found to be temporally expressed during gestation with the highest expression in early gestation and gradually decreasing expression as gestation progressed,the group previously explored its effect on WTAP expression.(1)LIN28B was found to be positively correlated with WTAP expression levels in early-onset pre-eclamptic tissues,and the expression correlation curves were constructed.(2)LIN28B inhibited WTAP expression through the high expression of Let-7a-2-3p axis in early-onset preeclampsia on WTAP expression let-7a-2-3p by"mi RNA targeting target gene".(3)LIN28B overexpression,co-transformed let-7a-2-3p,let-7a-2-3p was able to inhibit the positive regulation of WTAP by LIN28B.(3)HTR-8/SVneo,SWAN-71 cell lines,knockdown of WTAP downregulation was able to inhibit trophoblast proliferation,infiltration,and migration ability.HTR-8/SVneo cell lines RNA The results of the combined analysis of sequencing and MeRIP sequencing.(1)A total of 132 genes with decreased mRNA m~6A methylation level and 2812 genes with absolute decrease in mRNA m~6A methylation were identified in the knockdown HTR-8/SVneo cell lines tested;32 genes with increased mRNA m~6A methylation level and 507 genes with absolute increase in mRNA m~6A methylation were found.A total of 507 genes with increased mRNA m~6A methylation level,104 genes with decreased mRNA m~6A methylation level and absolute amount of mRNA m~6A methylation,including PEG10,RAB18,MCM5,NAXD,FZD2,etc.were found.(2)RNA-seq and MeRIP-seq association analysis showed that 45 expressions were down-regulated and 3 expressions were up-regulated among 104transcripts with both decreased mRNA m~6A methylation level and absolute mRNA m~6A methylation.(3)GSEA gene enrichment was found to be significantly reduced in knockdown HTR-8/SVneo cell lines with down-regulated WNT binding protein gene set enrichment.(4)Combined with the pre-eclampsia public database,FZD2 was found to be most significantly down-regulated in transcripts with decreased mRNA m~6A methylation levels and absolute mRNA m~6A methylation,and identified as a candidate molecule.(5)FZD2 was found to be positively regulated by WTAP in trophoblast HTR-8/SVneo and SWAN-71 cell lines.mRNA stability was significantly reduced after FZD2 knockdown by WTAP,and FZD2 downregulation was able to inhibit the proliferation,infiltration,and migration ability of trophoblast cells.(4)WTAP-regulated m~6A modification effects in preeclamptic rats with RUPP eclampsia In vivo validation.(1)RUPP pre-eclamptic rats were constructed,and Wtap and Fzd2 expression was significantly reduced in rat placenta.(2)After E14.5 placental fiber injection of overexpression Wtap lentivirus,the expression of Wtap gene in the placenta of overexpressed RUPP model rats could significantly improve the symptoms of pre-eclampsia in rats.Conclusions:(1)The level of total RNA m~6A methylation modification in early-onset preeclampsia placental tissues was lower than that in the control group,and there was a link between the change in m~6A methylation modification level and the onset of early-onset preeclampsia.WTAP down-regulation as a major methyltransferase was involved in the regulation of m~6A methylation modification level of total RNA.(2)WTAP was regulated in trophoblast cells by the LIN28B/let-7 axis in trophoblast cells,and the involvement of RNA m~6A may fluctuate during placental development.(3)The combined MeRIP-seq and RNA-seq analysis revealed that WTAP expression was down-regulated by the enrichment of"WNT binding protein",in which FZD2,a key gene,was involved.(4)In the placenta of RUPP model rats,Wtap and Fzd2 expression was lower than that of normal pregnant rats.Local overexpression of Wtap gene in the placenta significantly improved the symptoms of preeclampsia in rats.WTAP may play an important role in the pathogenesis and diagnosis of early-onset preeclampsia.