METTL3调控lncRNA的m6A修饰在衰老心肌缺血后处理自噬水平降低中的机制研究

The decrease of autophagy level in aged myocardium is an important mechanism of ischemic post-conditioning (IPO) against I/R injury, but the mechanism is unclear. lncRNA can regulate gene expression through a variety of mechanisms, and RNA m6A modification can affect the biological function of lncRNA. Our preliminary research indicated that lncRNA-AL627309 was a specific lncRNAs of decreased autophagy level of IPO in aged myocardium. Therefore, it is conjectured that METTL3 mediates lncRNA-AL627309 m6A modification regulated the autophagy level decreased, which is an important mechanism for IPO against I/R in aged myocardium. In order to verify this hypothesis, a specific lncRNA(lncRNA-AL627309) in the decreased autophagy level of aging myocardium was screened and confirmed, and the role in regulating the decreased autophagy level were discussed. Western blot was used to detect the changes of lncRNA-AL627309 m6a modification, and METTL3 was identified as the key regulatory enzyme. By using miCLIP directional detected the changes of specific lncRNA loci (191, 497) m6A modification, and to reveal the mechanism of METTL3 regulates lncRNA-AL627309 m6A modification and affects the binding of ATG13 promoter region in decreased autophagy levels of IPO in aged myocardium, providing a potential drug target for aging myocardial ischemia/reperfusion injury.

衰老心肌自噬水平降低是缺血后处理抗缺血/再灌注损伤的重要机制，但机制未清。lncRNA可通过多种机制调控基因表达，而m6A修饰可影响lncRNA的生物学功能。课题组预实验提示lncRNA-AL627309是衰老心肌IPO自噬水平降低的特异性lncRNA。故推测：METTL3调控lncRNA-AL627309m6A修饰是引起衰老心肌自噬水平下降的重要机制。为了验证该假说，筛选并确定衰老心肌自噬水平降低中的特异性lncRNA并予以验证，探讨其在调控自噬水平降低中的作用；采用免疫印迹等检测lncRNA-AL627309m6A修饰及调控因子的变化，明确METTL3为关键调控酶；利用miCLIP定向检测特异性lncRNA位点(191、497)m6A修饰的变化，揭示METTL3调控其m6A修饰进而影响与ATG13启动子区结合介导衰老心肌自噬水平降低的机制，为衰老心肌缺血/再灌注损伤提供潜在的药靶。

衰老心肌自噬水平降低是缺血后处理抗缺血/再灌注损伤的重要机制，但机制未清。lncRNA可通过多种机制调控基因表达，而m6A修饰可影响lncRNA的生物学功能。但特异性lncRNA的m6A修饰在衰老心肌自噬水平降低中的作用尚未有研究者涉及。基于以上背景，我们在研究中复制衰老心肌IPO模型，利用Western blot等实验检测其自噬水平变化，明确自噬在衰老心肌IPO中的作用。RNA测序等技术筛选并确定缺血后处理引起衰老心肌自噬水平下降的特异性lncRNA。采用免疫印迹等检测lncRNA m6A修饰及调控因子的变化，明确METTL3为关键调控酶；利用MeRIP-qPCR检测特异性lncRNA位点(191、497) m6A修饰的变化，揭示METTL3调控其m6A修饰介导衰老心肌自噬水平降低的机制。研究结果显示：在动物/细胞模型中，与假手术组或正常氧组相比，I/R组衰老心肌组织或H/R组衰老心肌细胞自噬水平明显升高；与I/R组或H/R组相比，IPostC组衰老心肌组织或HPostC组衰老心肌细胞自噬水平明显降低。测序结果显示lncRNA-AL627309是IPostC引起衰老心肌细胞自噬水平降低的特异性lncRNA。进一步研究发现lncRNA-AL627309 m6A修饰水平的改变会影响衰老心肌细胞自噬；构建lncRNA-AL627309 m6A单和/或双位点突变体，发现lncRNA-AL627309 191/497 双位点m6A修饰是衰老心肌IPostC自噬水平降低的机制。同时发现METTLE3参与lncRNA-AL627309 m6A修饰的调控，干扰METTLE3，发现lncRNA-AL627309 m6A水平明显降低，细胞自噬水平降低，揭示METTL3调控lncRNA-AL627309的m6A修饰是在衰老心肌缺血后处理自噬水平降低的机制，可以为衰老心肌缺血/再灌注损伤提供潜在的药靶。