{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,24]],"date-time":"2026-04-24T13:31:38Z","timestamp":1777037498722,"version":"3.51.4"},"reference-count":43,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,6,14]],"date-time":"2022-06-14T00:00:00Z","timestamp":1655164800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell Dev. Biol."],"abstract":"<jats:p>N<jats:sup>6<\/jats:sup>-methyladenosine (m<jats:sup>6<\/jats:sup>A) was the most abundant modification of mRNA and lncRNA in mammalian cells and played an important role in many biological processes. However, whether m<jats:sup>6<\/jats:sup>A modification was associated with recurrent spontaneous abortion (RSA) and its roles were still unclear.<\/jats:p><jats:p><jats:bold>Methods:<\/jats:bold> Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was used to study the global m<jats:sup>6<\/jats:sup>A modification pattern in RSAs and controls. RNA sequencing (RNA-Seq) was used to study the level of global mRNA in two groups. Real-time quantitative PCR (RT-qPCR) was used to verify the level of mRNA of METTL3 and ZBTB4. MeRIP\u2013qPCR was conducted to test the level of ZBTB4 m<jats:sup>6<\/jats:sup>A modification in two groups. In order to further explore whether ZBTB4 was the substrate of METTL3, the HTR-8\/SVneo (HTR-8) cell line was selected for the knockdown and overexpression of METTL3. To study whether METTL3 regulated the ZBTB4 expression by recognizing ZBTB4 mRNA m<jats:sup>6<\/jats:sup>A motifs in coding sequences (CDS), dual-luciferase reporter assay was conducted. RNA stability assays using actinomycin D were conducted to study the RNA stability of the HTR-8 cell line with METTL3 overexpression and knockdown. To illustrate the role of METTL3 in the invasion of trophoblast, matrigel invasion assays and transwell migration assays were conducted using the HTR-8 cell line with METTL3 overexpression and knockdown.<\/jats:p><jats:p><jats:bold>Results:<\/jats:bold> A total of 65 genes were found with significant differences both in m<jats:sup>6<\/jats:sup>A modification and mRNA expression. We found m<jats:sup>6<\/jats:sup>A methyltransferase METTL3 was significantly down-regulated in the RSA group. Through gene function analysis, RT-qPCR, MeRIP\u2013qPCR validation experiment, knockdown, and overexpression of METTL3 in the HTR-8 cell line, ZBTB4 was selected as one target of METTL3. Furthermore, we clarified that METTL3 regulated the expression of ZBTB4 by recognizing ZBTB4 mRNA m<jats:sup>6<\/jats:sup>A motifs in the CDS using the dual-luciferase reporter assay and METTL3 regulated the invasion of trophoblast by altering the stability and expression of ZBTB4 by RNA stability, matrigel invasion, and transwell migration assays.<\/jats:p><jats:p><jats:bold>Conclusion:<\/jats:bold> Our study revealed the mechanism by which METTL3 regulated the stability and expression of ZBTB4 and the trophoblast migration ability of RSA. A new perspective was provided for exploring the mechanism of embryonic development in RSA patients.<\/jats:p>","DOI":"10.3389\/fcell.2022.894810","type":"journal-article","created":{"date-parts":[[2022,6,14]],"date-time":"2022-06-14T07:09:45Z","timestamp":1655190585000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":26,"title":["METTL3-Mediated m6A RNA Methylation of ZBTB4 Interferes With Trophoblast Invasion and Maybe Involved in RSA"],"prefix":"10.3389","volume":"10","author":[{"given":"Nana","family":"Huang","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yue","family":"Gao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mengting","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liangjie","family":"Guo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Litao","family":"Qin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shixiu","family":"Liao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hongdan","family":"Wang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2022,6,14]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1016\/j.gde.2017.06.006","article-title":"The N6-Methyladenosine RNA Modification in Pluripotency and Reprogramming","volume":"46","author":"Aguilo","year":"2017","journal-title":"Curr. Opin. Genet. Dev."},{"key":"B2","doi-asserted-by":"publisher","first-page":"355","DOI":"10.1007\/s10815-017-1108-y","article-title":"Genetic and Epigenetic Variations Associated with Idiopathic Recurrent Pregnancy Loss","volume":"35","author":"Arias-Sosa","year":"2018","journal-title":"J. Assist. Reprod. Genet."},{"key":"B3","doi-asserted-by":"publisher","first-page":"48","DOI":"10.3389\/fpls.2012.00048","article-title":"Adenosine Methylation in Arabidopsis mRNA Is Associated with the 3\u2032 End and Reduced Levels Cause Developmental Defects","volume":"3","author":"Bodi","year":"2012","journal-title":"Front. Plant Sci."},{"key":"B4","doi-asserted-by":"publisher","first-page":"858","DOI":"10.1111\/jog.14250","article-title":"Association between Chronic Endometritis and Uterine Natural Killer Cell Density in Women with Recurrent Miscarriage: Clinical Implications","volume":"46","author":"Chen","year":"2020","journal-title":"J. Obstet. Gynaecol. Res."},{"key":"B5","doi-asserted-by":"publisher","first-page":"487","DOI":"10.1016\/j.omtn.2020.12.001","article-title":"METTL3-mediated m6A Modification of ZBTB4 mRNA Is Involved in the Smoking-Induced EMT in Cancer of the Lung","volume":"23","author":"Cheng","year":"2021","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"B6","doi-asserted-by":"publisher","first-page":"1131","DOI":"10.1158\/1940-6207.capr-12-0076-t","article-title":"An EMILIN1-Negative Microenvironment Promotes Tumor Cell Proliferation and Lymph Node Invasion","volume":"5","author":"Danussi","year":"2012","journal-title":"Cancer Prev. Res."},{"key":"B7","doi-asserted-by":"publisher","first-page":"152667","DOI":"10.1016\/j.prp.2019.152667","article-title":"MicroRNA-301b-3p Accelerates the Growth of Gastric Cancer Cells by Targeting Zinc Finger and BTB Domain Containing 4","volume":"215","author":"Fan","year":"2019","journal-title":"Pathology - Res. Pract."},{"key":"B8","doi-asserted-by":"publisher","first-page":"9684703","DOI":"10.1155\/2019\/9684703","article-title":"The lncRNA SOX2OT Rs9839776 C>T Polymorphism Indicates Recurrent Miscarriage Susceptibility in a Southern Chinese Population","volume":"2019","author":"Fang","year":"2019","journal-title":"Mediat. Inflamm."},{"key":"B9","doi-asserted-by":"publisher","first-page":"1002","DOI":"10.1126\/science.1261417","article-title":"Stem Cells. m6A mRNA Methylation Facilitates Resolution of Na\u00efve Pluripotency toward Differentiation","volume":"347","author":"Geula","year":"2015","journal-title":"Science"},{"key":"B10","doi-asserted-by":"publisher","first-page":"717832","DOI":"10.3389\/fimmu.2021.717832","article-title":"Key Gene and Functional Pathways Identified in Unexplained Recurrent Spontaneous Abortion Using Targeted RNA Sequencing and Clinical Analysis","volume":"12","author":"Gu","year":"2021","journal-title":"Front. Immunol."},{"key":"B11","doi-asserted-by":"publisher","first-page":"285","DOI":"10.1038\/s41556-018-0045-z","article-title":"Recognition of RNA N6-Methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation","volume":"20","author":"Huang","year":"2018","journal-title":"Nat. Cell Biol."},{"key":"B12","doi-asserted-by":"publisher","first-page":"e1007412","DOI":"10.1371\/journal.pgen.1007412","article-title":"Nuclear m6A Reader YTHDC1 Regulates Alternative Polyadenylation and Splicing during Mouse Oocyte Development","volume":"14","author":"Kasowitz","year":"2018","journal-title":"PLoS Genet."},{"key":"B13","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1095\/biolreprod.102.014977","article-title":"Endovascular Trophoblast Invasion: Implications for the Pathogenesis of Intrauterine Growth Retardation and Preeclampsia","volume":"69","author":"Kaufmann","year":"2003","journal-title":"Biol. Reprod."},{"key":"B14","doi-asserted-by":"publisher","first-page":"985","DOI":"10.2217\/epi-2016-0173","article-title":"Hypermethylated CpG Sites in the MTR Gene Promoter in Preterm Placenta","volume":"9","author":"Khot","year":"2017","journal-title":"Epigenomics"},{"key":"B15","doi-asserted-by":"publisher","first-page":"1034","DOI":"10.1038\/onc.2011.296","article-title":"Identification of Oncogenic microRNA-17-92\/ZBTB4\/specificity Protein axis in Breast Cancer","volume":"31","author":"Kim","year":"2012","journal-title":"Oncogene"},{"key":"B16","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1111\/j.1600-0897.2004.00247.x","article-title":"Regulation and Signal Transduction of Toll-like Receptors in Human Chorioncarcinoma Cell Lines","volume":"53","author":"Klaffenbach","year":"2005","journal-title":"Am. J. Reprod. Immunol."},{"key":"B17","doi-asserted-by":"publisher","first-page":"8640","DOI":"10.1038\/s41598-019-44714-1","article-title":"Functional Roles of hnRNPA2\/B1 Regulated by METTL3 in Mammalian Embryonic Development","volume":"9","author":"Kwon","year":"2019","journal-title":"Sci. Rep."},{"key":"B18","doi-asserted-by":"publisher","first-page":"444","DOI":"10.1038\/cr.2017.10","article-title":"Cytoplasmic m6A Reader YTHDF3 Promotes mRNA Translation","volume":"27","author":"Li","year":"2017","journal-title":"Cell Res."},{"key":"B19","doi-asserted-by":"publisher","first-page":"3853","DOI":"10.7150\/thno.31868","article-title":"The m6A Demethylase ALKBH5 Controls Trophoblast Invasion at the Maternal-Fetal Interface by Regulating the Stability of CYR61 mRNA","volume":"9","author":"Li","year":"2019","journal-title":"Theranostics"},{"key":"B20","doi-asserted-by":"publisher","first-page":"187","DOI":"10.1016\/j.fertnstert.2019.09.001","article-title":"Evaluation of Peripheral and Uterine Immune Status of Chronic Endometritis in Patients with Recurrent Reproductive Failure","volume":"113","author":"Li","year":"2020","journal-title":"Fertil. Steril."},{"key":"B21","doi-asserted-by":"publisher","first-page":"335","DOI":"10.1016\/j.molcel.2016.03.021","article-title":"The M 6 A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells","volume":"62","author":"Lin","year":"2016","journal-title":"Mol. Cell"},{"key":"B22","doi-asserted-by":"publisher","first-page":"93","DOI":"10.1038\/nchembio.1432","article-title":"A METTL3-METTL14 Complex Mediates Mammalian Nuclear RNA N6-Adenosine Methylation","volume":"10","author":"Liu","year":"2014","journal-title":"Nat. Chem. Biol."},{"key":"B23","doi-asserted-by":"publisher","first-page":"6051","DOI":"10.1093\/nar\/gkx141","article-title":"N 6-methyladenosine Alters RNA Structure to Regulate Binding of a Low-Complexity Protein","volume":"45","author":"Liu","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"B24","doi-asserted-by":"publisher","first-page":"1179","DOI":"10.1096\/fj.201800719r","article-title":"Mettl14 Is Required for Mouse Postimplantation Development by Facilitating Epiblast Maturation","volume":"33","author":"Meng","year":"2019","journal-title":"FASEB J."},{"key":"B25","doi-asserted-by":"publisher","first-page":"1635","DOI":"10.1016\/j.cell.2012.05.003","article-title":"Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3\u2032 UTRs and Near Stop Codons","volume":"149","author":"Meyer","year":"2012","journal-title":"Cell"},{"key":"B26","doi-asserted-by":"publisher","first-page":"874","DOI":"10.1016\/j.molcel.2016.02.027","article-title":"Codon Usage and 3\u2032 UTR Length Determine Maternal mRNA Stability in Zebrafish","volume":"61","author":"Mishima","year":"2016","journal-title":"Mol. Cell"},{"key":"B27","doi-asserted-by":"publisher","first-page":"177","DOI":"10.1038\/cr.2014.3","article-title":"Mammalian WTAP Is a Regulatory Subunit of the RNA N6-Methyladenosine Methyltransferase","volume":"24","author":"Ping","year":"2014","journal-title":"Cell Res."},{"key":"B28","doi-asserted-by":"publisher","first-page":"1187","DOI":"10.1016\/j.cell.2017.05.045","article-title":"Dynamic RNA Modifications in Gene Expression Regulation","volume":"169","author":"Roundtree","year":"2017","journal-title":"Cell"},{"key":"B29","doi-asserted-by":"publisher","first-page":"62","DOI":"10.1158\/0008-5472.can-16-1181","article-title":"Loss of the Methyl-CpG-Binding Protein ZBTB4 Alters Mitotic Checkpoint, Increases Aneuploidy, and Promotes Tumorigenesis","volume":"77","author":"Roussel-Gervais","year":"2017","journal-title":"Cancer Res."},{"key":"B30","doi-asserted-by":"publisher","first-page":"159","DOI":"10.1111\/j.1600-0897.2010.00837.x","article-title":"Placental Apoptosis in Health and Disease","volume":"64","author":"Sharp","year":"2010","journal-title":"Am. J. Reprod. Immunol."},{"key":"B31","doi-asserted-by":"publisher","first-page":"247","DOI":"10.1387\/ijdb.140083ms","article-title":"Adaptive Mechanisms Controlling Uterine Spiral Artery Remodeling during the Establishment of Pregnancy","volume":"58","author":"Soares","year":"2014","journal-title":"Int. J. Dev. Biol."},{"key":"B32","doi-asserted-by":"publisher","first-page":"e2004880","DOI":"10.1371\/journal.pbio.2004880","article-title":"METTL3-mediated m6A Modification Is Required for Cerebellar Development","volume":"16","author":"Wang","year":"2018","journal-title":"PLoS Biol."},{"key":"B33","doi-asserted-by":"publisher","first-page":"240","DOI":"10.1186\/s13023-020-01492-8","article-title":"Cloning, Expression and Enzyme Activity Delineation of Two Novel CANT1 Mutations: the Disappearance of Dimerization May Indicate the Change of Protein Conformation and Even Function","volume":"15","author":"Wang","year":"2020","journal-title":"Orphanet J. Rare Dis."},{"key":"B34","doi-asserted-by":"publisher","first-page":"e9880","DOI":"10.7717\/peerj.9880","article-title":"Integrated Analysis of the Transcriptome-wide m6A Methylome in Preeclampsia and Healthy Control Placentas","volume":"8","author":"Wang","year":"2020","journal-title":"PeerJ"},{"key":"B35","doi-asserted-by":"publisher","first-page":"651","DOI":"10.1038\/s41556-019-0315-4","article-title":"The RNA N(6)-methyladenosine Modification Landscape of Human Fetal Tissues","volume":"21","author":"Xiao","year":"2019","journal-title":"Nat. Cell Biol."},{"key":"B36","doi-asserted-by":"publisher","first-page":"1100","DOI":"10.1038\/cr.2017.100","article-title":"Mettl3-mediated m6A Regulates Spermatogonial Differentiation and Meiosis Initiation","volume":"27","author":"Xu","year":"2017","journal-title":"Cell Res."},{"key":"B37","doi-asserted-by":"publisher","first-page":"317","DOI":"10.1038\/s41586-021-03210-1","article-title":"METTL3 Regulates Heterochromatin in Mouse Embryonic Stem Cells","volume":"591","author":"Xu","year":"2021","journal-title":"Nature"},{"key":"B38","doi-asserted-by":"publisher","first-page":"616","DOI":"10.1038\/s41422-018-0040-8","article-title":"Dynamic Transcriptomic m6A Decoration: Writers, Erasers, Readers and Functions in RNA Metabolism","volume":"28","author":"Yang","year":"2018","journal-title":"Cell Res."},{"key":"B39","doi-asserted-by":"publisher","first-page":"108","DOI":"10.1016\/j.biopha.2018.01.132","article-title":"Tumor Suppressive ZBTB4 Inhibits Cell Growth by Regulating Cell Cycle Progression and Apoptosis in Ewing Sarcoma","volume":"100","author":"Yu","year":"2018","journal-title":"Biomed. Pharmacother."},{"key":"B40","doi-asserted-by":"publisher","first-page":"100973","DOI":"10.1016\/j.jbc.2021.100973","article-title":"The Detection and Functions of RNA Modification m6A Based on m6A Writers and Erasers","volume":"297","author":"Zhang","year":"2021","journal-title":"J. Biol. Chem."},{"key":"B41","doi-asserted-by":"publisher","first-page":"475","DOI":"10.1038\/nature21355","article-title":"m6A-dependent Maternal mRNA Clearance Facilitates Zebrafish Maternal-To-Zygotic Transition","volume":"542","author":"Zhao","year":"2017","journal-title":"Nature"},{"key":"B42","first-page":"2156","article-title":"Development and Validation of a m6A RNA Methylation Regulators-Based Signature for Predicting the Prognosis of Head and Neck Squamous Cell Carcinoma","volume":"9","author":"Zhao","year":"2019","journal-title":"Am. J. Cancer Res."},{"key":"B43","doi-asserted-by":"publisher","first-page":"18","DOI":"10.1016\/j.molcel.2012.10.015","article-title":"ALKBH5 Is a Mammalian RNA Demethylase that Impacts RNA Metabolism and Mouse Fertility","volume":"49","author":"Zheng","year":"2013","journal-title":"Mol. Cell"}],"container-title":["Frontiers in Cell and Developmental Biology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcell.2022.894810\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,6,14]],"date-time":"2022-06-14T07:09:50Z","timestamp":1655190590000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcell.2022.894810\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,14]]},"references-count":43,"alternative-id":["10.3389\/fcell.2022.894810"],"URL":"https:\/\/doi.org\/10.3389\/fcell.2022.894810","relation":{},"ISSN":["2296-634X"],"issn-type":[{"value":"2296-634X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,14]]},"article-number":"894810"}}