钙化性主动脉瓣疾病中m6A甲基化谱及表观调控机制

The m6A methylation profile and epigenetic regulatory mechanism in calcific aortic valve disease

摘要:

目的]构建钙化性主动脉瓣疾病(CAVD)的首个m⁶A甲基化谱,揭示m⁶A修饰在瓣膜钙化过程中的表观调控机制。 [方法]通过RNA甲基化测序(MeRIP-seq)技术获取CAVD患者瓣膜组织的m⁶A甲基化谱,运用生物信息学方法进行差异甲基化分析、GO功能注释及KEGG通路富集分析,筛选关键信号通路,以转化生长因子β(TGF-β)信号通路富集最显著；利用主动脉瓣膜间质细胞(AVIC)构建体外细胞钙化模型,采用RT-qPCR及Western blot检测肾母细胞瘤1相关蛋白(WTAP)、甲基转移酶样蛋白3(METTL3)、脂肪量和肥胖相关蛋白(FTO)、烷基化碱基修复蛋白同源物5(ALKBH5)、YTH结构域家族蛋白1/2(YTHDF1/2)等m⁶A甲基化关键酶,筛选核心调节因子；采用siRNA技术敲低ALKBH5,检测ALKBH5缺失对AVIC钙化及TGF-β信号通路的作用,并通过ALKBH5过表达实验进行双向验证,明确ALKBH5的作用；通过MeRIP-RT-qPCR对m⁶A修饰的具体通路靶基因SMAD家族成员2(SMAD2)进行甲基化验证。 [结果]通过MeRIP-seq成功获得了CAVD的首个m⁶A甲基化谱,发现钙化瓣膜组织m⁶A甲基化修饰水平显著升高(P＜0.01),并识别出7 412个m⁶A差异甲基化峰及TGF-β、丝裂原活化蛋白激酶(MAPK)、自噬等大量钙化相关的关键信号通路。体外AVIC钙化、ALKBH5敲低及过表达等实验证实,钙化AVIC中去甲基化酶ALKBH5表达下调(P＜0.05),ALKBH5敲低可以促进AVIC钙化,过表达则抑制其钙化。进一步机制研究发现,ALKBH5敲低后,钙化AVIC细胞中TGF-βⅡ型受体(TGFBR2)蛋白表达及SMAD2的磷酸化(p-SMAD2)水平均上调(均P＜0.05),TGF-β信号通路激活；另外,ALKBH5敲低后,SMAD2基因的m⁶A甲基化水平升高(P＜0.05)。 [结论]m⁶A甲基化修饰参与CAVD瓣膜钙化进程,去甲基化酶ALKBH5表达下调可能通过激活TGF-β信号通路,促进瓣膜间质细胞钙化。

Abstract:

Aim To establish the first m⁶A methylation profile for calcific aortic valve disease (CAVD) and elucidate the epigenetic regulatory mechanism of m⁶A modification during the process of valve calcification. Methods The m⁶A methylation profile of the valve tissues from CAVD patients was obtained through RNA methylation sequencing (MeRIP-seq). Differential methylation analysis, GO functional annotation, and KEGG pathway enrichment analysis were performed using bioinformatics methods to identify key signaling pathways, with the transforming growth factor-β (TGF-β) pathway showing the most significant enrichment. An in vitro calcification model using aortic valve interstitial cells (AVIC) was established, and RT-qPCR and Western blot analysis were employed to detect key m⁶A methylation enzymes, including Wilms' tumor 1-associating protein (WTAP), methyltransferase-like 3 (METTL3), fat mass and obesity-associated protein (FTO), alkylated base repair protein homolog 5 (ALKBH5), and YTH domain family proteins 1/2 (YTHDF1/2), to identify core regulatory factors. siRNA technology was used to knock down ALKBH5, and the effects of ALKBH5 deficiency on AVIC calcification and the TGF-β signaling pathway were detected. The results were further verified through ALKBH5 overexpression experiments, clearly demonstrating the role of ALKBH5. The methylation of specific pathway target gene mothers against decapentaplegic homolog 2 (SMAD2) was verified through MeRIP-RT-qPCR. Results The first m⁶A methylation profile of CAVD was successfully obtained through MeRIP-seq. It was found that the m⁶A methylation modification level in calcified valve tissues was significantly increased (P＜0.01), and 7 412 m⁶A differentially methylated peaks and a large number of key signaling pathways related to calcification, such as TGF-β, mitogen-activated protein kinase (MAPK), and autophagy, were identified. In vitro experiments on AVIC calcification, ALKBH5 knockdown, and overexpression confirmed that the expression of the demethylase ALKBH5 was downregulated in calcified AVIC (P＜0.05). ALKBH5 knockdown promoted AVIC calcification, while overexpression inhibited it. Further mechanism studies revealed that after ALKBH5 knockdown, the expression of TGF-β type Ⅱ receptor (TGFBR2) protein and the phosphorylation level of SMAD2 (p-SMAD2) in calcified AVIC were both upregulated (both P＜0.05), and the TGF-β signaling pathway was activated. Additionally, after ALKBH5 knockdown, the m⁶A methylation level of the SMAD2 gene increased (P＜0.05). Conclusions The m⁶A methylation modification is involved in the calcification process of CAVD valve. The downregulation of the demethylase ALKBH5 may promote the calcification of valve interstitial cells by activating the TGF-β signaling pathway.