Genetic and pharmacological inhibition of METTL3 alleviates renal fibrosis by reducing EVL m6A modification through an IGF2BP2-dependent mechanism.
Genetic and pharmacological inhibition of METTL3 alleviates renal fibrosis by reducing EVL m6A modification through an IGF2BP2-dependent mechanism.
复制标题
通过IGF2BP2依赖性机制减少EVL m6A修饰，对胃L3的遗传和药理学抑制可减轻肾纤维化。
DOI：
10.1002/ctm2.1359
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发表时间：
2023-08
影响因子：
10.6
通讯作者：
Meng, Xiao-Ming
中科院分区：
文献类型：
--
作者：
Ni, Wei-Jian;Zhou, Hong;Lu, Hao;Ma, Nan-Nan;Hou, Bing-Bing;Li, Wei;Kong, Fan-Xu;Yu, Ju-Tao;Hou, Rui;Jin, Juan;Wen, Jia-Gen;Zhang, Tao;Meng, Xiao-Ming
关键词：
N-6-甲基腺苷（m6 A）在肾脏生理和疾病进展中具有重要作用，但其在肾纤维化中的作用及机制尚待深入研究。因此，本研究将探索肾纤维化过程中关键调节因子介导的m6 A修饰的功能和潜在机制，从而探索有前景的抗肾纤维化药物。使用来自人类和小鼠的肾组织以及HK-2细胞作为研究对象。使用蛋白质印迹、定量真实的时间聚合酶链反应和其他方法在蛋白质和RNA水平上分析肾纤维化中m6 A修饰和调节剂的概况。采用甲基化RNA免疫沉淀测序和RNA测序结合甲基转移酶样3（stomach L3）条件性敲除的方法，探讨stomach L3的功能和潜在靶点。进行基因沉默和过表达结合RNA免疫沉淀，以研究胃L3调节促进肾纤维化的Ena/VASP样（EVL）m6 A修饰的潜在机制。本研究采用分子对接和虚拟筛选相结合的方法，结合体内外实验，筛选具有应用前景的中药单体，并探讨其调控胃L3/EVL m6 A轴和抗肾纤维化的作用机制。在纤维化肾脏的肾小管区域和HK-2细胞中，胃L3和m6 A修饰被过度激活。上调的胃L3增强了EVL mRNA的m6 A修饰，以胰岛素样生长因子2 mRNA结合蛋白2（IGF 2BP 2）依赖性方式改善其稳定性和表达。高表达的EVL与Smad 7的结合消除了Smad 7诱导的对转化生长因子-β（TGF-β1）/Smad 3信号转导的抑制，这反过来促进了肾纤维化的进展。通过分子对接和虚拟筛选，从中药中鉴定出一种名为异甘草苷的单体，该单体可抑制中药中的L3活性，同时抑制L3/EVL m6 A轴发挥抗肾纤维化作用。总的来说，过度激活的胃L3/EVL m6 A轴是肾纤维化治疗的潜在靶点，异甘草硫苷对胃L3活性的药理学抑制表明它是一种有前途的抗肾纤维化药物。纤维化前刺激过度激活TEC和肾组织中的MELTT 3和m6 A修饰。上调的胃L3通过IGF 2BP 2依赖性机制增强EVL mRNA的m6 A修饰。EVL与Smad 7结合以减弱其对TGF-β1/Smad 3的抑制，这反过来促进肾纤维化。抑制胃L3和胃L3/EVL m6 A轴可显著减轻肾纤维化。异甘草苷是一种潜在的抗纤维化药物，可抑制胃L3/EVL m6 A轴。
N 6‐methyladenosine (m6A) is of great importance in renal physiology and disease progression, but its function and mechanism in renal fibrosis remain to be comprehensively and extensively explored. Hence, this study will explore the function and potential mechanism of critical regulator‐mediated m6A modification during renal fibrosis and thereby explore promising anti‐renal fibrosis agents. Renal tissues from humans and mice as well as HK‐2 cells were used as research subjects. The profiles of m6A modification and regulators in renal fibrosis were analysed at the protein and RNA levels using Western blotting, quantitative real‐time polymerase chain reaction and other methods. Methylation RNA immunoprecipitation sequencing and RNA sequencing coupled with methyltransferase‐like 3 (METTL3) conditional knockout were used to explore the function of METTL3 and potential targets. Gene silencing and overexpression combined with RNA immunoprecipitation were performed to investigate the underlying mechanism by which METTL3 regulates the Ena/VASP‐like (EVL) m6A modification that promotes renal fibrosis. Molecular docking and virtual screening with in vitro and in vivo experiments were applied to screen promising traditional Chinese medicine (TCM) monomers and explore their mechanism of regulating the METTL3/EVL m6A axis and anti‐renal fibrosis. METTL3 and m6A modifications were hyperactivated in both the tubular region of fibrotic kidneys and HK‐2 cells. Upregulated METTL3 enhanced the m6A modification of EVL mRNA to improve its stability and expression in an insulin‐like growth factor 2 mRNA‐binding protein 2 (IGF2BP2)‐dependent manner. Highly expressed EVL binding to Smad7 abrogated the Smad7‐induced suppression of transforming growth factor‐β (TGF‐β1)/Smad3 signal transduction, which conversely facilitated renal fibrosis progression. Molecular docking and virtual screening based on the structure of METTL3 identified a TCM monomer named isoforsythiaside, which inhibited METTL3 activity together with the METTL3/EVL m6A axis to exert anti‐renal fibrosis effects. Collectively, the overactivated METTL3/EVL m6A axis is a potential target for renal fibrosis therapy, and the pharmacological inhibition of METTL3 activity by isoforsythiaside suggests that it is a promising anti‐renal fibrosis agent. Prefibrotic stimuli overactivate MELTT3 and m6A modifications in TECs and renal tissues. Upregulated METTL3 enhances the m6A modification of EVL mRNA through an IGF2BP2‐dependent mechanism. EVL binds to Smad7 to attenuate its inhibition of TGF‐β1/Smad3, which conversely promotes renal fibrosis. Inhibition of METTL3 and the METTL3/EVL m6A axis markedly alleviates renal fibrosis. Isoforsythiaside is a potential antifibrotic agent that inhibits the METTL3/EVL m6A axis.
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