Department of Pharmacy, Anhui Provincial Hospital, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, People's Republic of China.
Inflammation and Immune Mediated Diseases Laboratory of Anhui Province, Anhui Institute of Innovative Drugs, School of Pharmacy, Anhui Medical University, Hefei, Anhui, People's Republic of China.
Clin Transl Med. 2023 Aug;13(8):e1359. doi: 10.1002/ctm2.1359.
N -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.
N-甲基腺苷（m6A）在肾脏生理和疾病进展中非常重要，但它在肾纤维化中的功能和机制仍有待全面广泛地探索。因此，本研究将探讨关键调节因子介导的 m6A 修饰在肾纤维化过程中的功能和潜在机制，从而探索有前途的抗肾纤维化药物。
以人肾组织、小鼠肾组织和 HK-2 细胞为研究对象。采用 Western blot、实时定量聚合酶链反应等方法分析肾纤维化中 m6A 修饰和调节因子在蛋白质和 RNA 水平上的特征。采用甲基化 RNA 免疫沉淀测序和 RNA 测序与甲基转移酶样 3（METTL3）条件性敲除相结合的方法，探讨 METTL3 的功能及其潜在靶点。通过基因沉默和过表达结合 RNA 免疫沉淀，研究 METTL3 调节 Ena/VASP 样（EVL）m6A 修饰促进肾纤维化的潜在机制。通过体外和体内实验进行分子对接和虚拟筛选，筛选有前途的中药单体并探讨其调节 METTL3/EVL m6A 轴和抗肾纤维化的机制。
METTL3 和 m6A 修饰在纤维化肾脏的肾小管区域和 HK-2 细胞中均过度激活。上调的 METTL3 通过胰岛素样生长因子 2 mRNA 结合蛋白 2（IGF2BP2）依赖性方式增强 EVL mRNA 的 m6A 修饰，提高其稳定性和表达。高表达的 EVL 与 Smad7 结合，消除了 Smad7 诱导的转化生长因子-β（TGF-β1）/Smad3 信号转导抑制，反而促进了肾纤维化的进展。基于 METTL3 结构的分子对接和虚拟筛选鉴定出一种中药单体名为异连翘脂苷，它通过抑制 METTL3 活性以及 METTL3/EVL m6A 轴发挥抗肾纤维化作用。
总之，过度激活的 METTL3/EVL m6A 轴是肾纤维化治疗的潜在靶点，异连翘脂苷抑制 METTL3 活性提示其是一种有前途的抗肾纤维化药物。
