Inhibition of METTL3/m6A/miR126 promotes the migration and invasion of endometrial stromal cells in endometriosis†.

N 6-methyladenosine (m6A), one of the most abundant RNA modifications, is involved in the progression of many diseases, but its role and related molecular mechanisms in endometriosis remain unknown. To address these issues, we detected m6A levels in normal, eutopic, and ectopic endometrium and found the m6A levels decreased in eutopic and ectopic endometrium compared with normal endometrium. In addition, we proved that methyltransferase-like 3 (METTL3) downregulation accounted for m6A reduction in endometriosis. Furthermore, we observed that METTL3 knockdown facilitated the migration and invasion of human endometrial stromal cells (HESCs), whereas METTL3 overexpression exerted opposite effects, suggesting that METTL3 downregulation might contribute to endometriosis development by enhancing cellular migration and invasion. Mechanistically, METTL3-dependent m6A was involved in the DGCR8-mediated maturation of primary microRNA126 (miR126 and pri-miR126). Moreover, miR126 inhibitor significantly enhanced the migration and invasion of METTL3-overexpressing HESCs, whereas miR126 mimics attenuated the migration and invasion of METTL3-silenced HESCs. Our study revealed the METTL3/m6A/miR126 pathway, whose inhibition might contribute to endometriosis development by enhancing cellular migration and invasion. It also showed that METTL3 might be a novel diagnostic biomarker and therapeutic target for endometriosis.

Li X ，Xiong W ，Long X ，Dai X ，Peng Y ，Xu Y ，Zhang Z ，Zhang L ，Liu Y ... -  《-》

METTL3 is aberrantly expressed in endometriosis and suppresses proliferation, invasion, and migration of endometrial stromal cells.

Endometriosis (EM) is one of the leading gynecological disorders, and associated with excessive functioning of endometrial stromal cells (ESCs). The current study was conducted to determine the expression and role of methyltransferase-like 3 (METTL3) in the proliferation, invasion, and migration of ESCs in EM. The documented expression levels of METTL3, microRNA (miR)-21-5p, and WNT inhibitory factor 1 (WIF1) in eutopic (Eut) and ectopic (Ect) endometrial tissues and ESCs were determined by a combination of real-time quantitative polymerase chain reaction and Western blot assay. After transfection with pcDNA3.1-METTL3, miR-21-5p mimic, and WIF1 small interfering RNA, cell counting kit-8, colony formation, and Transwell assays were performed in the Ect ESCs (Ect-ESCs). Subsequently, the binding of miR-21-5p to METTL3 was analyzed, along with quantification of the N6-methyladenosine (m6A) level, the enrichments of METTL3 and m6A on WIF1, and the mRNA stability of WIF1. In our findings, METTL3 was downregulated in the EM tissues and cells. METTL3 overexpression intrinsically reduced the proliferation, invasion, and migration of Ect-ESCs. miR-21-5p inhibited the METTL3 expression while METTL3 enhanced the mRNA stability and expression of WIF1 via m6A modification. Additionally, a negative correlation of METTL3 was identified with miR-21-5p along with a positive correlation with the WIF1 mRNA in EM tissues. The miR-21-5p overexpression or WIF1 downregulation enhanced the proliferation, invasion, and migration of Ect-ESCs. Collectively, miR-21-5p inhibited the METTL3-mediated m6A modification and mRNA stability of WIF1, thereby facilitating the proliferation, invasion, and migration of Ect-ESCs.

Zhang QC 《-》

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

Xiong W ，Jin J ，Liu Y 《-》

MicroRNA-126-5p downregulates BCAR3 expression to promote cell migration and invasion in endometriosis.

Endometriosis (EMs) is an estrogen-dependent multifactorial disease. Inhibition of estrogen in endometrial cells contributes to their failure to form lesions in ectopic sites. However, whether reducing or suppressing the inhibitory effect of estrogen results in the establishment of ectopic lesions remains unclear. The BCAR3 gene induces estrogen resistance in estrogen-dependent breast cancer cells and promotes cell migration, invasion, and epithelial-mesenchymal transition (EMT). However, the expression of BCAR3 in endometriosis and its effect on endometrial cell function and the anti-estrogen effect of endometriosis have not been reported. These issues are addressed in the present study. The study included 32 cases of ectopic endometrium and eutopic endometrium in patients with endometriosis and 31 cases of normal endometrium as controls. The expression of BCAR3 and microRNA (miR)-126-5p was detected by real-time PCR, immunohistochemistry, and western blotting. The effects of BCAR3 and miR-126-5p on the morphology and biological behavior of eutopic endometrial cells were verified using lentivirus overexpression and a vector knockdown model, the CCK-8 assay, Transwell experiments, and estrogen intervention experiments using primary cultures of epithelial and stromal cells. The BCAR3 gene was highly expressed in ectopic endometrium and the eutopic endometrium of patients with endometriosis, and the expression level was higher in stage III-IV patients than in stage I-II patients. In vitro cell experiments showed that miR-126-5p negatively regulated the expression of BCAR3 and its effect on the migration and invasion of stromal cells. Low expression of miR-126-5p and high expression of BCAR3 promoted endometriosis stromal cell migration and invasion. Assessment of EMT in endometriosis compared with eutopic endometrium showed that the expression of vimentin was significantly increased and the expression of E-cadherin was significantly decreased in ectopic endometrium. Estrogen promoted EMT in eutopic endometrial epithelial cells and this effect was reversed by estrogen inhibitors. BCAR3 had no direct effect on EMT and did not act synergistically with estrogen on promoting EMT. miR-126-5p negatively regulated BCAR3 expression in eutopic endometriosis, enhanced the migration and invasion of endometrial cells, and promoted the occurrence of endometriosis. BCAR3 did not induce EMT and had no synergistic effect with estrogen, but its inhibition of anti-estrogen function may provide new insight into the mechanism of local estrogen action in endometriosis.

Meng X ，Liu J ，Wang H ，Chen P ，Wang D ... -  《-》