Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China.
Mol Med. 2023 Feb 6;29(1):19. doi: 10.1186/s10020-023-00604-x.
With the increasing morbidity and mortality of preeclampsia (PE), it has posed a huge challenge to public health. Previous studies have reported endoplasmic reticulum (ER) stress could contribute to trophoblastic dysfunction which was associated with the N-methyladenosine (mA) modification by methyltransferase-like 3 (METTL3), resulting in PE. However, little was known about the relationship between METTL3 and ER stress in PE. Thus, in vitro and in vivo studies were performed to clarify the mechanism about how METTL3 affects the trophoblasts under ER stress in PE and to explore a therapeutic approach for PE.
An ER stress model in HTR-8/SVneo cells and a preeclamptic rat model were used to study the mechanism and explore a therapeutic approach for PE. Western blot, immunohistochemistry, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and methylated RNA immunoprecipitation (MeRIP)-qPCR were performed to detect the protein, RNA, and methylated transmembrane BAX inhibitor motif containing 6 (TMBIM6) expression levels. The mA colorimetric and mRNA stability assays were used to measure the mA levels and TMBIM6 stability, respectively. Short hairpin RNAs (shRNAs) were used to knockdown METTL3 and YTH N6-methyladenosine RNA binding protein 2 (YTHDF2). Flow cytometry and Transwell assays were performed to evaluate the apoptosis and invasion abilities of trophoblasts.
Upregulated METTL3 and mA levels and downregulated TMBIM6 levels were observed in preeclamptic placentas under ER stress. The ER stress model was successfully constructed, and knockdown of METTL3 had a beneficial effect on HTR-8/SVneo cells under ER stress as it decreased the levels of methylated TMBIM6 mRNA. Moreover, overexpression of TMBIM6 was beneficial to HTR-8/SVneo cells under ER stress as it could neutralize the harmful effects of METTL3 overexpression. Similar to the knockdown of METTL3, downregulation of YTHDF2 expression resulted in the increased expression and mRNA stability of TMBIM6. Finally, improved systemic symptoms as well as protected placentas and fetuses were demonstrated in vivo.
METTL3/YTHDF2/TMBIM6 axis exerts a significant role in trophoblast dysfunction resulting in PE while inhibiting METTL3 may provide a novel therapeutic approach for PE.
子痫前期（PE）的发病率和死亡率不断上升，对公共健康构成了巨大挑战。先前的研究表明，内质网（ER）应激可导致滋养层功能障碍，这与甲基转移酶样 3（METTL3）介导的 N-甲基腺苷（mA）修饰有关，从而导致 PE。然而，对于 METTL3 与 PE 中的 ER 应激之间的关系知之甚少。因此，进行了体外和体内研究，以阐明 METTL3 在 ER 应激下如何影响滋养层细胞的机制，并探索治疗 PE 的方法。
使用 ER 应激模型的 HTR-8/SVneo 细胞和子痫前期大鼠模型来研究机制并探索治疗 PE 的方法。进行 Western blot、免疫组织化学、定量逆转录聚合酶链反应（qRT-PCR）和甲基化 RNA 免疫沉淀（MeRIP）-qPCR 来检测蛋白质、RNA 和甲基化跨膜 BAX 抑制剂基序包含 6（TMBIM6）的表达水平。mA 比色法和 mRNA 稳定性测定分别用于测量 mA 水平和 TMBIM6 稳定性。短发夹 RNA（shRNA）用于敲低 METTL3 和 YTH N6-甲基腺苷 RNA 结合蛋白 2（YTHDF2）。流式细胞术和 Transwell 测定用于评估滋养层细胞的凋亡和侵袭能力。
在 ER 应激下，子痫前期胎盘组织中观察到 METTL3 和 mA 水平上调，TMBIM6 水平下调。成功构建了 ER 应激模型，并且敲低 METTL3 对 ER 应激下的 HTR-8/SVneo 细胞具有有益作用，因为它降低了甲基化 TMBIM6 mRNA 的水平。此外，过表达 TMBIM6 对 ER 应激下的 HTR-8/SVneo 细胞有益，因为它可以中和 METTL3 过表达的有害作用。与敲低 METTL3 相似，下调 YTHDF2 表达导致 TMBIM6 的表达和 mRNA 稳定性增加。最后，在体内观察到全身症状改善以及胎盘和胎儿得到保护。
METTL3/YTHDF2/TMBIM6 轴在导致 PE 的滋养层功能障碍中发挥重要作用，而抑制 METTL3 可能为 PE 提供一种新的治疗方法。
