Aims Calcific aortic valve disease (CAVD) is a common heart valve disease with significant clinical consequences. The mechanisms that drive the pathogenesis of CAVD remain to be fully elucidated. N6-methyladenosine (m6A), the most prevalent RNA epigenetic regulator, has recently been implicated in cardiovascular disease, but its role in CAVD has yet to be investigated. In this study, we investigated the potential function of m6A modification in CAVD. Methods and results Using clinical samples from CAVD patients in combination with human valve interstitial cell (hVIC) calcification model, we screened the expression of m6A modulators and discovered that alkB homolog 5, RNA demethylase (ALKBH5), a key m6A demethylase, was significantly down-regulated in calcified hVICs and human aortic valves. Consistently, increased m6A levels were seen in calcified hVICs, and treatment with 3-deazaadenosine (DAA), an inhibitor of m6A modification, significantly reduced hVIC osteogenic differentiation and calcification. In addition, we showed that silencing of ALKBH5 expression increased global m6A levels and accelerated hVIC osteogenic differentiation and calcification, whereas overexpression of ALKBH5 resulted in the opposite effect. We demonstrated that ALKBH5 directly modulate m6A levels of TGFBR2 and its mRNA stability, leading to altered TGFBR2 expression and SMAD2 signalling in hVICs. We further showed that inhibition of TGFBR2 or knockdown of SMAD2 attenuated ALKBH5 knockdown-induced hVIC osteogenic differentiation and calcification. The expression of the m6A reader protein YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) was up-regulated during the process of hVIC calcification. Intriguingly, we revealed that the ALKBH5 silencing-induced increased hVIC osteogenic differentiation and calcification were abolished after knockdown of YTHDF1. These data suggest a potential role YTHDF1 in aortic valve calcification. Conclusion This study showed that ALKBH5 attenuated aortic valve calcification through the TGFBR2/SMAD2 signalling pathway via direct m6A modification of TGFBR2.

中文翻译：

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N6-甲基腺苷脱甲基酶 ALKBH5 是主动脉瓣钙化的新型表观遗传调节因子


钙化性主动脉瓣病 （CAVD） 是一种常见的心脏瓣膜疾病，具有严重的临床后果。驱动 CAVD 发病机制的机制仍有待完全阐明。N6-甲基腺苷 （m6A） 是最普遍的 RNA 表观遗传调节因子，最近与心血管疾病有关，但其在 CAVD 中的作用尚未得到研究。在这项研究中，我们研究了 m6A 修饰在 CAVD 中的潜在功能。方法和结果 使用 CAVD 患者的临床样本结合人瓣膜间质细胞 （hVIC） 钙化模型，我们筛选了 m6A 调节剂的表达，发现 alkB 同源物 5，RNA 脱甲基化酶 （ALKBH5），一种关键的 m6A 脱甲基化酶，在钙化 hVICs 和人主动脉瓣中显着下调。一致地，在钙化 hVICs 中观察到 m6A 水平升高，并且用 m6A 修饰抑制剂 3-脱氮腺苷 （DAA） 治疗可显着减少 hVIC 成骨分化和钙化。此外，我们发现 ALKBH5 表达的沉默增加了整体 m6A 水平并加速了 hVIC 成骨分化和钙化，而 ALKBH5 的过表达导致相反的效果。我们证明 ALKBH5 直接调节 TGFBR2 的 m6A 水平及其 mRNA 稳定性，导致 hVICs 中 TGFBR2 表达和 SMAD2 信号传导改变。我们进一步表明，抑制 TGFBR2 或敲低 SMAD2 减弱了 ALKBH5 敲除诱导的 hVIC 成骨分化和钙化。在 hVIC 钙化过程中，m6A reader 蛋白 YTH N6-甲基腺苷 RNA 结合蛋白 F1 （YTHDF1） 的表达上调。 有趣的是，我们揭示了 ALKBH5 沉默诱导的 hVIC 成骨分化和钙化增加在敲低 YTHDF1 后被消除。这些数据表明 YTHDF1 在主动脉瓣钙化中可能起作用。结论 本研究显示，ALKBH5 通过 TGFBR2 的直接 m6A 修饰通过 TGFBR2/SMAD2 信号通路减轻主动脉瓣钙化。