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 experiments. For this purpose, HT-22 cells were treated with hemin to mimic ICH , 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.

中文翻译：

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METTL3 沉默抑制 NEK7 的 m6A 甲基化，从而抑制基于 HT-22 细胞的脑出血模型中的热解

脑出血（ICH）会引起严重的神经损伤，其进展是由 METTL3 驱动的。本研究旨在通过实验探讨METTL3在ICH中的作用。为此，用氯化血红素处理 HT-22 细胞以模拟 ICH，然后使用流式细胞术、乳酸脱氢酶释放分析、酶联免疫吸附测定和蛋白质印迹评估细胞焦亡。此外，使用甲基化 RNA 免疫沉淀、RNA 免疫沉淀、双荧光素酶报告基因测定和定量实时聚合酶链反应评估 NEK7 的 N6-甲基腺苷 (m6A) 甲基化。结果表明，METTL3 的敲低可抑制氯高铁血红素诱导的细胞焦亡，并由于 METTL3 下调而抑制 NEK7 的 m6A 甲基化，从而降低 NEK7 mRNA 的稳定性。过表达 NEK7 可以消除对 METTL3 诱导的细胞焦亡的影响，而 IGF2BP2 则增加 NEK7 的表达。同样，IGF2BP2 沉默下调 METTL3 介导的 NEK7 表达。总之，沉默 METTL3 通过抑制 IGF2BP2 识别的 NEK7 m6A 甲基化来抑制氯化血红素诱导的 HT-22 细胞焦亡。这些发现有望确定一种新的脑出血治疗策略。