LncRNAH19启动子区域DNA甲基化调控VSMCs表型转化及其在动脉钙化中的作用机制

Arterial calcification(AC) is one of the most important clinical indicators of cardio-cerebrovascular disease(CCVD). It also plays a crucial role in the pathological mechanism of atherosclerotic diseases and closely associated with phenotypic transformation of VSMCs. Research has shown that long non-coding RNA H19 stimulates the differentiation of osteoblasts by inhibiting NOTCH1 signal pathway and participate in osteogenesis,however the mechanism of H19 in arterial calcification is not clear. Preliminary result of our earlier research shows a higher level of lncRNA H19 in patients with arterial calcification compared with individuals without AC . In light of this, we hypothesize that “Dysregulation of DNA methylation in the promoter region of H19 during AC promotes the phenotypic transformation of VSMCs and initiates osteogenic process by NOTCH1-HEY1-RUNX2 pathway”. Our project is to measure the DNA methylation in the promoter region of H19 in AC patients to determine the correlation between H19 and AC, construct AC model in vivo and in vitro,using siRNA technology, to illuminate the mechanism of the DNA methylation in the promoter region of H19 to promote the phenotypic transformation of VSMCs and osteogenic process by NOTCH1 pathway. The implementation of the projects will provide new ideas for prophylaxis and treatment of adverse cardiovascular events mediated by AC.

动脉钙化是临床上一项重要的心脑血管疾病预警指标，是动脉粥样硬化性疾病的重要病理机制之一，与VSMCs的成骨样表型转化密切相关。研究表明，lncRNA H19通过抑制NOTCH1信号通路促进成骨细胞分化，参与骨组织形成，但其在动脉钙化中的作用机制尚不明确。我们前期试验结果表明，动脉钙化患者lncRNA H19表达水平明显高于健康对照者。基于此，我们提出假说：lncRNA H19启动子区域低甲基化通过NOTCH1-HEY1-RUNX2信号通路促进VSMCs表型转化及动脉钙化的发生发展。本项目拟通过测量动脉钙化患者lncRNA H19启动子区域DNA甲基化水平，明确其与动脉钙化相关性；构建在体及离体钙化模型，采用siRNA干扰等技术，阐明H19启动子区域DNA甲基化通过NOTCH1信号通路在VSMCs表型转化及钙化发生发展中的分子机制。项目的实施将为临床防治动脉钙化导致的心血管事件提供新思路。

背景及目的：动脉钙化(AC)主要是由血管平滑肌细胞(VSMCs)成骨表型转化引起的。长链非编码RNA H19 (lncRNA H19)因其重要的转录调控能力而日益受到关注。我们报道了lncRNA H19在VSMCs转化后表达上调。因此，我们旨在研究H19在VSMCs转变中的作用及其分子机制。.内容及方法：用10mmβ-甘油磷酸诱导 HA-VSMCs 发生表型转化，通过qPCR和Western Blot分析检测细胞内成骨相关蛋白RUNX2及平滑肌标志物 α-SMA 的表达水平，通过茜素红s染色及碱性磷酸酶ALP含量测定实验检测钙化水平，通过检测泛素化连接蛋白 LC-3、P62 观察自噬水平变化。.结果：与正常VSMCs相比，钙化的VSMCs中lncRNA H19、Runx2和OSX的表达均显著升高，而分化标志物SM22-α和α- sma的表达则显著降低。SiRNA研究显示，敲低lncRNA H19可降低VSMCs钙化和Runx2的表达。我们进一步验证了lncRNA H19通过p38 MAPK和ERK1/2信号转导通路促进VSMCs钙化。与正常组相比，钙化诱导 HA-VSMCs组 lncＲNA H19 表达量明显升高，成骨相关蛋白 ＲUNX2 表达水平增加，平滑肌标志物 α-SMA 表达下调，自噬被抑制，同时MAPK信号通路关键因子p-p38和p-p44/42的表达增加。敲低 lncRNA H19 后，平滑肌细胞表型转化减缓，钙化程度减弱，自噬水平有所回升，p-p38和p-p44/42的表达下调。.结论：lncRNA H19促进VSMCs的转化，其机制可能与抑制VSMCs 的保护性自噬以及激活MAPK/ Runx2轴有关。这一发现不仅揭示了lncRNA H19的新功能，也为lncRNA H19参与Runx2调控通路在AC中的作用提供了新的观点，并可为AC的早期诊断和治疗提供新的指示。