METHODS: HPH model and pulmonary artery smooth muscle cells (PASMCs) were constructed from which m6A changes were observed and screened for AlkB homolog 5 (Alkbh5). Alkbh5 knock-in (KI) and knock-out (KO) mice were constructed to observe the effects on m6A and evaluate right ventricular systolic pressure (RVSP), left ventricular and septal weight [RV/(LV + S)], and pulmonary vascular remodeling in the context of HPH. Additionally, the effects of Alkbh5 knockdown using adenovirus were examined in vitro on m6A, specifically in PASMCs with regard to proliferation, migration and cytochrome P450 1A1 (Cyp1a1) mRNA stability.
RESULTS: In both HPH mice lung tissues and hypoxic PASMCs, a decrease in m6A was observed, accompanied by a significant up-regulation of Alkbh5 expression. Loss of Alkbh5 attenuated the proliferation and migration of hypoxic PASMCs in vitro, with an associated increase in m6A modification. Furthermore, Alkbh5 KO mice exhibited reduced RVSP, RV/(LV + S), and attenuated vascular remodeling in HPH mice. Mechanistically, loss of Alkbh5 inhibited Cyp1a1 mRNA decay and increased its expression through an m6A-dependent post-transcriptional mechanism, which hindered the proliferation and migration of hypoxic PASMCs.
CONCLUSIONS: The current study highlights the loss of Alkbh5 impedes the proliferation and migration of PASMCs by inhibiting post-transcriptional Cyp1a1 mRNA decay in an m6A-dependent manner.
方法:构建HPH模型和肺动脉平滑肌细胞(PASMC),观察m6A变化,筛选AlkB同源物5(Alkbh5)。构建Alkbh5敲入(KI)和敲除(KO)小鼠,以观察其对m6A的影响并评估右心室收缩压(RVSP),左心室和间隔重量[RV/(LV+S)],和HPH背景下的肺血管重塑。此外,在体外检查了使用腺病毒的Alkbh5敲低对m6A的影响,特别是在PASMC中,关于增殖,迁移和细胞色素P4501A1(Cyp1a1)mRNA的稳定性。
结果:在HPH小鼠肺组织和低氧PASMC中,观察到M6A下降,伴随着Alkbh5表达的显著上调。Alkbh5的丢失在体外减弱了低氧PASMCs的增殖和迁移,与M6A修饰的相关增加。此外,Alkbh5KO小鼠表现出降低的RVSP,RV/(LV+S),并减弱HPH小鼠的血管重塑。机械上,Alkbh5的缺失抑制了Cyp1a1mRNA的衰减,并通过m6A依赖性转录后机制增加了其表达,这阻碍了低氧PASMC的增殖和迁移。
结论:当前的研究强调了Alkbh5的缺失通过以m6A依赖性方式抑制转录后Cyp1a1mRNA衰减来阻碍PASMCs的增殖和迁移。