miR-214对血管钙化的调控作用及其机制研究

Vascular calcification is an active, cell-regulated process. Phenotypic transition/dedifferentiation of mature smooth muscle cells (SMCs) into an osteogenic phenotype is a key feature in vascular calcification. MicroRNAs (miRNAs) are a large class of evolutionarily conserved, small, endogenous, noncoding RNAs serving as essential post-transcriptional modulators of gene expression. In vivo and in vitro study showed that miRNAs regulate vascular calcification by targeting ostegenic transcription factors. However, knowledge of miRNA biology in cardiovascular calcification is still very limited. Our previous study showed that miR-214 has a crucial role in suppressing bone formation by directly targeting ATF4 to inhibit osteoblast activity. Real time PCR also showed that level of circulating miR-214 decreased in the serum of coronary calcification patients. In vitro studied showed ATF4 expression co-related with the level of miR-214, thus provide the clue that ATF4 is a possible osteogenic transcription target for miR-214. In this proposed research, through mouse VSMC osteoblast differentiation induced by β-glycerophosphate and miR214 knock out mouse, the effects of miR-214 on regulating VSMC calcification will be investigated in detail, the effect of miRNA -214 regulating VSMC in targeting ATF4 during vascular calcification will be further elucidated through in-vitro and in vivo study. The therapeutic effect of miRNA 214 on vascular calcification will be also investigated by miRNA 214 agomir and Vit D treated mice. In summary, the objective of the research is to elucidate the underlying mechanism of miRNA 214 in the regulation of vascular calcification, thus providing a potential novel therapeutic target for vascular calcification.

血管钙化的主要特征是血管平滑肌细胞（VSMC）的迁移、增殖和骨向分化。miRNA是一种转录后水平调控蛋白表达的小RNA分子，参与骨调节的miRNA可通过调节骨相关因子影响血管钙化。我们前期证实miR-214通过靶向作用于ATF4抑制成骨细胞分化及骨形成。并且发现冠脉血管钙化病人血清中miR-214表达下降，体外培养VSMC中ATF4表达同miR-214明显相关。提示miR-214对ATF4的调控可能在血管钙化发生过程中发挥重要调控作用。本课题通过体外细胞学模型及在体小鼠血管钙化模型，利用miR-214 VSMC特异敲除小鼠研究miR-214同血管钙化之间的内在联系及变化规律，从细胞和整体水平上揭示miR-214对VSMC骨向分化调控的分子途径，探讨miR-214/ATF4调节通路在血管钙化中的作用,探索体内干预miR-214对抗血管钙化的可能性，从miRNA的角度寻找血管钙化新的治疗靶点。

本课题立在通过体外细胞学模型及在体小鼠血管钙化模型，利用miR-214 VSMC特异敲除小鼠研究miR-214同血管钙化之间的内在联系及变化规律，从细胞和整体水平上揭示miR-214对VSMC骨向分化调控的分子途径，探讨miR-214/ATF4调节通路在血管钙化中的作用,探索体内干预miR-214对抗血管钙化的可能性，从miRNA的角度寻找血管钙化新的治疗靶点。但在在试验的过程中发现，通过细胞试验证实通过平滑肌细胞培养，miR-214 并未能使其发生骨向分化，从而我们未能得出miR-214 影响钙化的结论。相反，我们发现miRNA对表型转化和增殖具有调控作用。收缩型血管平滑肌细胞中miR-214表达升高，而 增殖型血管平滑肌细胞中miR-214表达降低，从而提示miRNA 在平滑肌细胞中的独特作用。对此我们开展一系列离体研究，发现血管平滑肌细胞来源的miR-214对于血管收缩特性的维持有重要作用，可以作为血管平滑肌细胞表型转换的标志分子，体外实验证明miR-214对于血管收缩特性的维持有重要作用，miR-214在体外实验中抑制平滑肌细胞的增殖，在血管平滑肌细胞中PTEN可能为miR-214的靶基因。我们构建了miR-214全身敲除（miR-214 KO）小鼠，证实在生理条件下，全身敲除miR-214不影响小鼠的基础血压，但以增强小鼠的血管功能。在WT和KO小鼠体内埋缓释泵灌注AngII 28天造高血压模型，在生理条件下，平滑肌细胞特异性敲除miR-214不影响小鼠的基础血压，miR-214 CKO 敲除可以缓解AngII诱导的高血压，平滑肌细胞特异性敲除miR-214可以缓解AngII诱导的血管重塑。