Department of Gynaecology, The First Affiliated Hospital of Harbin Medical University, Harbin, China.
J Transl Med. 2023 Jun 23;21(1):407. doi: 10.1186/s12967-023-04209-0.
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.
子宫内膜异位症（EMs）是一种功能子宫内膜在子宫外的异位种植，是导致不孕和盆腔疼痛的主要原因。N6-甲基腺苷（m6A）作为一种基本的 mRNA 修饰，参与各种病理过程。然而，m6A RNA 修饰在子宫内膜异位症中的作用尚不清楚。本研究探讨了 METTL3 介导的 m6A 修饰及其在子宫内膜异位症中的作用机制。
采用 RT-PCR 分析 EMs 中的主要 m6A 调控因子。通过 m6A-mRNA 表转录组微阵列和 RNA 测序评估 METTL3 的候选靶标和可能的机制。采用原代子宫内膜干细胞（ESCs）模型验证 METTL3 对 SIRT1 mRNA m6A 修饰的影响，并通过 RT-PCR、Western blot、MeRIP 和 RIP 实验阐明机制。采用 CCK-8 细胞活力测定、Transwell 侵袭实验、EdU 增殖实验、划痕愈合迁移实验和衰老相关β-半乳糖苷酶染色实验研究 METTL3 和 SIRT1 在 ESCs 体外的潜在生物学机制。采用 PgrCre/ + METTL3-/- 雌性纯合子小鼠模型和裸鼠异种移植模型进一步研究 METTL3 介导的 m6A 改变对 EMs 的生理后果。
数据显示，METTL3 表达下调显著降低 ESCs 中 m6A RNA 甲基化水平。沉默 METTL3 介导的 m6A 修饰可促进 ESCs 在体外的增殖、迁移和侵袭。m6A 阅读器蛋白 YTHDF2 与 m6A 修饰结合，诱导 SIRT1 mRNA 的降解。随后抑制 SIRT1/FOXO3a 信号通路的激活，促进 ESCs 的细胞衰老，并抑制 ESCs 在体外和体内的异位种植。
研究结果表明，METTL3 介导的 m6A 甲基化通过表观遗传调控 ESCs 的异位种植，从而导致子宫内膜异位症的进展。本研究确立了 METTL3-YTHDF2-SIRT1/FOXO3a 作为子宫内膜异位症的关键轴和潜在机制。
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