METTL3-regulated m6A modification impairs the decidualization of endometrial stromal cells by regulating YTHDF2-mediated degradation of FOXO1 mRNA in endometriosis-related infertility.

Endometriosis-related infertility is a common worldwide reproductive health concern. Despite ongoing research, the causes of infertility remain unclear. Evidence suggests that epigenetic regulation is crucial in reproduction. However, the role of N6-methyladenosine (m6A) modification of RNA in endometriosis-related infertility requires further investigation. We examined the expression of m6A and methyltransferase-like 3 (METTL3) in endometrial samples taken from normal fertile women in the proliferative phase (the NP group) or the mid-secretory phase (the NS group) or from women with endometriosis-related infertility at the mid-secretory phase (the ES group). We treated primary endometrial stromal cells (ESCs) with medroxyprogesterone acetate and 8-Bromo-cyclic adenosine monophosphate for in vitro decidualization and detected the expression of m6A, METTL3, and decidual markers. We analyzed the expression of m6A, METTL3, and forkhead box O1 (FOXO1) in ESCs from normal fertile women (the ND group) or women with endometriosis-related infertility (the ED group). We also assessed the expression of m6A, METTL3, and decidual markers, as well as the embryo adhesion rate, upon METTL3 overexpression or knockdown. Additionally, we investigated the role of METTL3 in embryo implantation in vivo by applying mice with endometriosis. Furthermore, we performed RNA stability assays, RNA immunoprecipitation (RIP), and methylated RIP assays to explore the mechanisms underlying the regulation of FOXO1 by METTL3-mediated m6A. The expression of m6A and METTL3 was reduced only in the NS group; the NP and ES groups demonstrated increased m6A and METTL3 levels. m6A and METTL3 levels decreased in ESCs with prolonged decidual treatment. Compared to the ND group, m6A and METTL3 levels in the ED group increased after decidual treatment, whereas the expression of FOXO1 decreased. METTL3 overexpression suppressed the expression of decidual markers and embryo implantation in vitro; METTL3 knockdown exhibited the opposite effect. Inhibition of METTL3 promoted embryo implantation in vivo. Furthermore, we observed that METTL3-mediated m6A regulated the degradation of FOXO1 mRNA through YTHDF2, a m6A binding protein. METTL3-regulated m6A promotes YTHDF2-mediated decay of FOXO1 mRNA, thereby affecting cellular decidualization and embryo implantation. These findings provide novel insights into the development of therapies for women with endometriosis-related infertility.

Li X ，Jin J ，Long X ，Weng R ，Xiong W ，Liang J ，Liu J ，Sun J ，Cai X ，Zhang L ，Liu Y ... -  《Reproductive Biology and Endocrinology》

METTL3-mediated m6A modification of SIRT1 mRNA inhibits progression of endometriosis by cellular senescence enhancing.

Endometriosis (EMs), the ectopic planting of functional endometrium outside of the uterus, is a leading cause of infertility and pelvic pain. As a fundamental mRNA modification, N6-methyladenosine (m6A) participates in various pathological processes. However, the role of m6A RNA modification in endometriosis remains unclear. The present study explores METTL3-mediated m6A modification and the mechanisms involved in endometriosis. The dominant m6A regulators in EMs were analysed using RT‒PCR. Candidate targets and possible mechanisms of METTL3 were assessed by m6A-mRNA epitranscriptomic microarray and RNA sequencing. A primary ESCs model was employed to verify the effect of METTL3 on m6A modification of SIRT1 mRNA, and the mechanism was elucidated by RT‒PCR, Western blotting, MeRIP, and RIP assays. CCK-8 viability assays, Transwell invasion assays, EdU proliferation assays, wound healing migration assays, and senescence-associated β-galactosidase staining were performed to illuminate the potential biological mechanism of METTL3 and SIRT1 in ESCs in vitro. An in vivo PgrCre/ + METTL3 -/- female homozygous mouse model and a nude mouse xenograft model were employed to further investigate the physiologic consequences of METTL3-mediated m6A alteration on EMs. Our data show that decreased METTL3 expression significantly downregulates m6A RNA methylation levels in ESCs. Silencing m6A modifications mediated by METTL3 accelerates ESCs viability, proliferation, migration, and invasion in vitro. The m6A reader protein YTHDF2 binds to m6A modifications to induce the degradation of SIRT1 mRNA. SIRT1/FOXO3a signalling pathway activation is subsequently inhibited, promoting the cellular senescence of ESCs and inhibiting the ectopic implantation of ESCs in vitro and in vivo. Our findings demonstrate that METTL3-mediated m6A methylation epigenetically regulates the ectopic implantation of ESCs, resulting in the progression of endometriosis. Our study establishes METTL3-YTHDF2-SIRT1/FOXO3a as a critical axis and potential mechanism in endometriosis.

Wang X ，Wang J ，Zhao X ，Wu H ，Li J ，Cheng Y ，Guo Q ，Cao X ，Liang T ，Sun L ，Zhang G ... -  《Journal of Translational Medicine》

Scribble downregulation in adenomyosis compromises endometrial stromal decidualization by decreasing FOXO1 expression.

Does Scribble (SCRIB) contribute to aberrant decidualization of endometrial stromal cells (ESC) in adenomyosis? SCRIB knockdown impairs decidualization of ESC by decreasing Fork-head box O1A (FOXO1) expression through the protein kinase B (AKT) and atypical protein kinase C (aPKC) activated pathways. Stromal SCRIB is required for primary decidual zone formation and pregnancy success in mice. In our previous studies, decidualization was dampened in ESC isolated from adenomyosis patients, yet the underlying molecular mechanisms remain elusive. Eutopic endometrium tissue samples from diffuse adenomyosis and non-adenomyosis patients in proliferative, early-secretory and mid-secretory phase (n = 10 per phase for each group) were explored. In parallel, in vitro decidualization studies were carried out in ESC isolated from non-adenomyosis women (n = 8). The endometrial SCRIB expression was analyzed using immunohistochemistry staining and western blot. Quantitative RT-PCR (qRT-PCR), western blot and immunofluorescence staining were used to explore the expression of SCRIB in ESC during in vitro decidualization. siRNA-mediated SCRIB knockdown followed by decidual markers expression analysis, flow cytometry for cell cycle analysis and phalloidin staining for morphological analysis were performed to examine the function of SCRIB in ESC decidualization. RNA-sequencing was performed to examine the SCRIB-mediated transcriptional changes in decidualized ESC (DSC). Rescue experiments using an AKT inhibitor MK2206 and aPKC inhibitor NSC37044 were used to investigate the signaling pathways through which could mediate SCRIB-regulated FOXO1 protein expression and ESC decidualization. We found that the expression of SCRIB in the mid-secretory phase eutopic endometrial stroma of adenomyosis patients was significantly lower than that of non-adenomyosis. SCRIB knockdown reduced the expression of decidual markers, abrogated the epithelioid-like morphological changes, inhibited the mesenchymal-to-epithelial transitions process and promoted the cell cycle progression of ESC during in vitro decidualization. SCRIB knockdown-induced decidualization defects were attributed to a decrease in expression of transcription factor FOXO1, known to regulate decidualization. Furthermore, we found that SCRIB knockdown induced the aberrant activation of AKT and aPKC, which led to FOXO1 phosphorylation and degradation. Rescue assay confirmed that restoring the expression of FOXO1 effectively reversed the decidualization defects and cell cycle progression caused by SCRIB knockdown. N/A. In this study, it was demonstrated that SCRIB knockdown mediated the activation of AKT and aPKC, contributing to FOXO1 degradation and aberrant decidualization, however, the molecular link between AKT and aPKC signaling was not determined, and still requires further exploration. Our findings support the hypothesis that adenomyosis interferes with embryo implantation due to insufficient endometrial receptivity. Abnormal decidualization of the endometrial stroma may clarify the possible association between adenomyosis and infertility. Our findings may be clinically useful for counseling and treatment of infertile adenomyosis patients. This study was supported by the National Natural Science Foundation of China (82001523 and 82171639). The authors have no conflicts of interest to disclose. N/A.

Peng Y ，Liu X ，Jin Z ，Liu H ，Xu C ... -  《-》

METTL3-dependent m6A modification facilitates decreased endometrial decidualization via attenuation of MET in endometriosis.

Xiong W ，Jin J ，Liu Y 《-》

METTL3 Mediates Epithelial-Mesenchymal Transition by Modulating FOXO1 mRNA N(6) -Methyladenosine-Dependent YTHDF2 Binding: A Novel Mechanism of Radiation-Induced Lung Injury.

The biological roles of epithelial-mesenchymal transition (EMT) in the pathogenesis of radiation-induced lung injury (RILI) have been widely demonstrated, but the mechanisms involved have been incompletely elucidated. N6 -methyladenosine (m6 A) modification, the most abundant reversible methylation modification in eukaryotic mRNAs, plays vital roles in multiple biological processes. Whether and how m6 A modification participates in ionizing radiation (IR)-induced EMT and RILI remain unclear. Here, significantly increased m6 A levels upon IR-induced EMT are detected both in vivo and in vitro. Furthermore, upregulated methyltransferase-like 3 (METTL3) expression and downregulated α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) expression are detected. In addition, blocking METTL3-mediated m6 A modification suppresses IR-induced EMT both in vivo and in vitro. Mechanistically, forkhead box O1 (FOXO1) is identified as a key target of METTL3 by a methylated RNA immunoprecipitation (MeRIP) assay. FOXO1 expression is downregulated by METTL3-mediated mRNA m6 A modification in a YTH-domain family 2 (YTHDF2)-dependent manner, which subsequently activates the AKT and ERK signaling pathways. Overall, the present study shows that IR-responsive METTL3 is involved in IR-induced EMT, probably by activating the AKT and ERK signaling pathways via YTHDF2-dependent FOXO1 m6 A modification, which may be a novel mechanism involved in the occurrence and development of RILI.

Feng Y ，Yuan P ，Guo H ，Gu L ，Yang Z ，Wang J ，Zhu W ，Zhang Q ，Cao J ，Wang L ，Jiao Y ... -  《Advanced Science》