Abstract：
Intracerebral hemorrhage (ICH) induces severe neurological damage, and its progression is driven by METTL3. This study aimed to investigate the role of METTL3 in ICH via in vitro experiments. For this purpose, HT-22 cells were treated with hemin to mimic ICH in vitro, followed by evaluating cell pyroptosis using flow cytometry, lactic dehydrogenase release analysis, enzyme-linked immunosorbent assay, and western blotting. Moreover, N6-methyl adenosine (m6A) methylation of NEK7 was assessed using methylated RNA immunoprecipitation, RNA immunoprecipitation, dual-luciferase reporter assay, and quantitative real-time polymerase chain reaction. Results indicated that knockdown of METTL3 inhibited hemin-induced pyroptosis and suppressed m6A methylation of NEK7 due to METTL3 downregulation, reducing NEK7 mRNA stability. The effects on METTL3-induced cell pyroptosis were abrogated by overexpressing NEK7, while IGF2BP2 increased NEK7 expression. Similarly, IGF2BP2 silence downregulated NEK7 expression mediated by METTL3. In conclusion, silencing of METTL3 inhibited hemin-induced HT-22 cell pyroptosis by suppressing m6A methylation of NEK7, which was recognized by IGF2BP2. These findings are envisaged to identify a novel therapeutic strategy for ICH.
摘要：
脑出血（ICH）导致严重的神经损伤，它的发展是由METTL3驱动的。本研究旨在通过体外实验研究METTL3在ICH中的作用。为此,用血红素处理HT-22细胞以体外模拟ICH，然后用流式细胞术评估细胞焦亡，乳酸脱氢酶释放分析，酶联免疫吸附测定，和西方印迹。此外，使用甲基化RNA免疫沉淀评估NEK7的N6-甲基腺苷（m6A）甲基化，RNA免疫沉淀，双荧光素酶报告分析，和定量实时聚合酶链反应。结果表明，METTL3的敲低抑制了血红素诱导的焦凋亡，并抑制了METTL3下调导致的NEK7的m6A甲基化，降低NEK7mRNA稳定性。通过过表达NEK7消除了对METTL3诱导的细胞焦亡的影响，而IGF2BP2增加了NEK7的表达。同样，IGF2BP2沉默下调METTL3介导的NEK7表达。总之,METTL3的沉默通过抑制被IGF2BP2识别的NEK7的m6A甲基化来抑制血红素诱导的HT-22细胞的焦亡。这些发现旨在确定ICH的新治疗策略。
