Department of Anesthesiology, The First Hospital of China Medical University, Shenyang, China.
Stem Cell Res Ther. 2024 Feb 16;15(1):43. doi: 10.1186/s13287-024-03658-8.
Ischemia-reperfusion injury to the central nervous system often causes severe complications. The activation of endogenous neural stem cells (NSCs) is considered a promising therapeutic strategy for nerve repair. However, the specific biological processes and molecular mechanisms of NSC activation remain unclear, and the role of N6-methyladenosine (mA) methylation modification in this process has not been explored.
NSCs were subjected to hypoxia/reoxygenation (H/R) to simulate ischemia-reperfusion in vivo. mA RNA methylation quantitative kit was used to measure the total RNA mA methylation level. Quantitative real-time PCR was used to detect methyltransferase and demethylase mRNA expression levels. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were conducted for NSCs in control and H/R groups, and the sequencing results were analyzed using bioinformatics. Finally, the migration ability of NSCs was identified by wound healing assays, and the proliferative capacity of NSCs was assessed using the cell counting kit-8, EdU assays and cell spheroidization assays.
Overall of mA modification level and Mettl14 mRNA expression increased in NSCs after H/R treatment. The mA methylation and expression profiles of mRNAs in NSCs after H/R are described for the first time. Through the joint analysis of MeRIP-seq and RNA-seq results, we verified the proliferation of NSCs after H/R, which was regulated by mA methylation modification. Seven hub genes were identified to play key roles in the regulatory process. Knockdown of Mettl14 significantly inhibited the proliferation of NSCs. In addition, separate analysis of the MeRIP-seq results suggested that mA methylation regulates cell migration and differentiation in ways other than affecting mRNA expression. Subsequent experiments confirmed the migration ability of NSCs was suppressed by knockdown of Mettl14.
The biological behaviors of NSCs after H/R are closely related to mA methylation of mRNAs, and Mettl14 was confirmed to be involved in cell proliferation and migration.
中枢神经系统的缺血再灌注损伤常导致严重并发症。内源性神经干细胞（NSCs）的激活被认为是神经修复的一种有前途的治疗策略。然而，NSC 激活的具体生物学过程和分子机制尚不清楚，并且该过程中 N6-甲基腺苷（mA）甲基化修饰的作用尚未得到探索。
将 NSCs 进行缺氧/复氧（H/R）处理以模拟体内的缺血再灌注。使用 mA RNA 甲基化定量试剂盒测量总 RNA mA 甲基化水平。采用定量实时 PCR 检测甲基转移酶和去甲基化酶 mRNA 表达水平。对对照组和 H/R 组的 NSCs 进行甲基化 RNA 免疫沉淀测序（MeRIP-seq）和 RNA 测序（RNA-seq），并使用生物信息学进行分析。最后，通过划痕愈合实验鉴定 NSCs 的迁移能力，通过细胞计数试剂盒-8（CCK-8）、EdU 实验和细胞球体形成实验评估 NSCs 的增殖能力。
在 H/R 处理后，NSCs 中的整体 mA 修饰水平和 Mettl14 mRNA 表达增加。首次描述了 NSCs 经历 H/R 后 mA 甲基化和 mRNA 表达谱。通过 MeRIP-seq 和 RNA-seq 结果的联合分析，我们验证了 H/R 后 NSCs 的增殖是由 mA 甲基化修饰调节的。鉴定出 7 个关键基因在调节过程中发挥关键作用。敲低 Mettl14 显著抑制 NSCs 的增殖。此外，对 MeRIP-seq 结果的单独分析表明，mA 甲基化通过影响 mRNA 表达以外的方式调节细胞迁移和分化。后续实验证实，敲低 Mettl14 抑制了 NSCs 的迁移能力。
H/R 后 NSCs 的生物学行为与 mRNAs 的 mA 甲基化密切相关，并且证实 Mettl14 参与了细胞增殖和迁移。
