miR-34b/c整合启动子区甲基化和下游靶基因对动脉钙化的调控机制研究

Arterial calcification is common in patients with end-stage kidney disease and type 2 diabetes and results in significantly higher incidence of cardiovascular events. Recent studies have demonstrated that the changes in miRNA levels are relevant to the development of arterial calcification, and the abnormal status of promoter methylation is possibly one of important approaches in the aberrant regulation of miRNA. In our previous studies supported by the National Natural Science Foundation of China (30900622), we found that the expression of miR-34b/c is decreased in the arterial calcification tissue compared with normal tissue. Furthermore, there is an increased DNA methylation of the promoter region of miR-34 b/c with lower expression levels during the transition of vascular smooth muscle cells into osteoblast-like cells. Based on in-vitro cellular and in-vivo animal models of arterial calcification, the present study will systematically investigate possible mechanism of miR-34b/c, which is expected to be a core factor participating in arterial calcification, through epigenetic regulations focusing on both its upstream promoter methylation and downstream putative target genes (Satb2 and/or Runx2). This study aims at illustrating the role of miR-34 b/c and its promoter methylation on arterial calcification, and is possible to demonstrate a new theory for prevention and treatment on this disease.

动脉钙化常见于终末期肾病和2型糖尿病患者，导致心血管事件显著增高。近期研究显示，microRNA（miRNA）表达变化与动脉钙化的发生发展密切相关，启动子区甲基化异常可能是miRNA表达失调的重要调控方式。在国家自然科学基金资助下（项目编号：30900622），我们前期研究发现：与正常组织相比，动脉钙化组织中的miR-34b/c表达降低，血管平滑肌细胞向成骨样细胞分化过程中，miR-34b/c表达下降而其启动子区甲基化增强。本课题拟采用动脉钙化的体外细胞模型和体内动物模型，以miR-34b/c为研究核心，从miR-34b/c上游的启动子区甲基化和下游的靶基因（Satb2 和/或 Runx2）系统探讨miR-34b/c调控动脉钙化的作用机制，阐明miR-34b/c及其启动子区甲基化在动脉钙化调控过程中的作用。本项目的实施，将揭示动脉钙化新的发病机制，为动脉钙化防治提供新的作用靶点和理论依据。

动脉钙化是与慢性肾脏病、2型糖尿病、高血压和骨质疏松密切相关的一种疾病，与心血管事件的发生存在关联。研究表明，血管平滑肌细胞（VSMCs）向成骨样细胞分化是动脉钙化的关键机制。MiRNAs是一种内源性非编码小分子RNA，参与调节细胞增殖、分化、凋亡和迁移的过程。本项目采用β-甘油磷酸（β-GP）诱导VSMCs向成骨样细胞分化，成功构建体外钙化模型。我们发现miR-34b在VSMCs钙化过程中的表达受到抑制。进一步研究发现，过表达miR-34b可以降低Runx2和Satb2的蛋白表达并且抑制β-GP诱导的VSMCs钙化，而低表达miR-34b后可以增加Runx2和Satb2的表达并且增强β-GP诱导的VSMCs钙化，提示miR-34b通过靶向Runx2和Satb2参与VSMCs向成骨样细胞分化。亚硫酸氢钠PCR(BSP)发现，VSMCs成骨分化过程中miR-34b甲基化水平升高。低表达DNMT3a可以阻断β-GP引起的miR-34b甲基化，提示DNMT3a可以调节miR-34b的甲基化并参与VSMCs成骨分化。我们发现与正常者相比，尿毒症患者的肾动脉钙化明显增多,miR-34b表达降低，miR-34b启动子区甲基化水平明显升高，并且DNMT3a、Runx2和Satb2在尿毒症患者的肾动脉中表达也是增加的。最后，通过注射miR-34b agomirs体内过表达miR-34b可以抑制维生素D诱导的动脉钙化。通过本项目的实施，我们证实了miR-34b的抑制动脉钙化作用和作用机制，为动脉钙化的防治提供新的治疗靶点，同时为动脉钙化的防治方法开辟新的思路。.在项目的资助下，我们同时探讨了omentin对动脉钙化的影响及其机制。我们发现omentin 在体外抑制VSMCs成骨分化并且可以引起AMPK磷酸化。AMPK信号通路抑制剂可以阻断omentin的抑制VSMCs钙化作用，而AMPK信号通路激活剂可以进一步增强omentin的抑制VSMCs钙化作用。最后，我们在小鼠动脉钙化模型中证实omentin可以抑制动脉钙化而AMPK抑制剂可以部分阻断omentin的作用。这为动脉钙化的防治提供新的靶点。