BACKGROUND:Calcification of the aortic valve leads to increased leaflet stiffness and consequently results in the development of calcific aortic valve disease (CAVD). However, the underlying molecular and cellular mechanisms of calcification remain unclear. Here, we identified a novel aortic valve calcification-associated PIWI-interacting RNA (piRNA; AVCAPIR) that increases valvular calcification and promotes CAVD progression.METHODS:Using piRNA sequencing, we identified piRNAs contributing to the pathogenesis of CAVD that we termed AVCAPIRs. High-cholesterol diet–fed ApoE^–/– mice with AVCAPIR knockout were used to examine the role of AVCAPIR in aortic valve calcification (AVC). Gain- and loss-of-function assays were conducted to determine the role of AVCAPIR in the induced osteogenic differentiation of human valvular interstitial cells. To dissect the mechanisms underlying AVCAPIR-elicited procalcific effects, we performed various analyses, including an RNA pulldown assay followed by liquid chromatography-tandem mass spectrometry, methylated RNA immunoprecipitation sequencing, and RNA sequencing. RNA pulldown and RNA immunoprecipitation assays were used to study piRNA interactions with proteins.RESULTS:We found that AVCAPIR was significantly upregulated during AVC and exhibited potential diagnostic value for CAVD. AVCAPIR deletion markedly ameliorated AVC in high-cholesterol diet–fed ApoE^–/– mice, as shown by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased peak transvalvular jet velocity and mean transvalvular pressure gradient, as well as increased aortic valve area), and diminished levels of osteogenic markers (Runx2 and Osterix) in aortic valves. These results were confirmed in osteogenic medium–induced human valvular interstitial cells. Using unbiased protein-RNA screening and molecular validation, we found that AVCAPIR directly interacts with FTO (fat mass and obesity-associated protein), subsequently blocking its N⁶-methyladenosine demethylase activity. Further transcriptomic and N⁶-methyladenosine modification epitranscriptomic screening and molecular validation confirmed that AVCAPIR hindered FTO-mediated demethylation of CD36 mRNA transcripts, thus enhancing CD36 mRNA stability through the N⁶-methyladenosine reader IGF2BP1 (insulin-like growth factor 2 mRNA binding protein). In turn, the AVCAPIR-dependent increase in CD36 stabilizes its binding partner PCSK9 (proprotein convertase subtilisin/kexin type 9), a procalcific gene, at the protein level, which accelerates the progression of AVC.CONCLUSIONS:We identified a novel piRNA that induced AVC through an RNA epigenetic mechanism and provide novel insights into piRNA-directed theranostics in CAVD.

中文翻译：

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AVCAPIR：钙化主动脉瓣疾病中一种新型的促钙化 PIWI 相互作用 RNA

背景：主动脉瓣钙化导致小叶硬度增加，从而导致钙化主动脉瓣疾病（CAVD）的发生。然而，钙化的潜在分子和细胞机制仍不清楚。在这里，我们鉴定了一种新的主​​动脉瓣钙化相关 PIWI 相互作用 RNA (piRNA；AVCAPIR)，它会增加瓣膜钙化并促进 CAVD 进展。 方法：使用 piRNA 测序，我们鉴定了有助于 CAVD 发病机制的 piRNA，我们将其称为 AVCAPIR。使用高胆固醇饮食喂养的 AVCAPIR 敲除的 ApoE ^–/–小鼠来检查 AVCAPIR 在主动脉瓣钙化 (AVC) 中的作用。进行功能获得和功能丧失测定以确定 AVCAPIR 在诱导人瓣膜间质细胞成骨分化中的作用。为了剖析 AVCAPIR 引起的促钙化效应的机制，我们进行了各种分析，包括 RNA Pulldown 测定，然后进行液相色谱-串联质谱分析、甲基化 RNA 免疫沉淀测序和 RNA 测序。RNA Pulldown 和 RNA 免疫沉淀试验用于研究 piRNA 与蛋白质的相互作用。结果：我们发现 AVCAPIR 在 AVC 过程中显着上调，并对 CAVD 表现出潜在的诊断价值。AVCAPIR 缺失显着改善了高胆固醇饮食喂养的 ApoE ^–/–小鼠的 AVC，如主动脉瓣叶厚度和钙沉积减少、超声心动图参数改善（跨瓣峰值射流速度和平均跨瓣压力梯度降低以及主动脉瓣面积增加），主动脉瓣中成骨标志物（Runx2 和 Osterix）水平降低。这些结果在成骨培养基诱导的人瓣膜间质细胞中得到证实。通过无偏的蛋白质-RNA 筛选和分子验证，我们发现 AVCAPIR 直接与 FTO（脂肪量和肥胖相关蛋白）相互作用，随后阻断其N ⁶ -甲基腺苷脱甲基酶活性。进一步的转录组学和N ⁶ -甲基腺苷修饰表观转录组筛选和分子验证证实，AVCAPIR 阻碍了 FTO 介导的 CD36 mRNA 转录本的去甲基化，从而通过N ⁶ -甲基腺苷读取器 IGF2BP1（胰岛素样生长因子 2 mRNA 结合蛋白）增强 CD36 mRNA 的稳定性。反过来，CD36 的 AVCAPIR 依赖性增加在蛋白质水平上稳定了其结合伙伴 PCSK9（前蛋白转化酶枯草杆菌蛋白酶/kexin 9 型）（一种前钙化基因），从而加速了 AVC 的进展。结论：我们鉴定了一种新的 piRNA，可诱导AVC 通过 RNA 表观遗传机制，为 CAVD 中 piRNA 指导的治疗诊断提供新的见解。