circRNA_5303通过miR-138-5p调控Smad4参与钙化性主动脉瓣膜病变的分子机制研究

Calcific aortic valve disease can lead to serious cardiovascular complications, but its pathogenesis remains largely unclear. CircRNA plays an important role in the regulation of myocardial cell development, differentiation, proliferation and apoptosis, but the role of circRNA in the development of valve calcification is rarely studied. We found that the expression of circRNA_5303 and its downstream target gene Smad4 was significantly higher in calcified aortic valve, while the expression of miR-138-5p was significantly lower, indicating that circRNA_5303 may play a positive regulatory role in the development of active valve calcification. we will investigate the molecular mechanism of circRNA_5303 promoting the osteogenic differentiation of human aortic valve interstitial cells via miR-138-5p/Smad4, and further investigate whether the regulation of circRNA_5303 and miR-138-5p expression levels in hAVICs also exerted an effect in vivo. Our results suggest that circRNA_5303 might be a novel biomarker or therapeutic target for regulating osteoblast differentiation in calcific aortic valve disease. We hope our research could provide some new insight of clinical therapeutic for the patients with calcified aortic valvular disease.

钙化性主动脉瓣膜病会导致严重的心血管并发病，但其发病机制目前仍不十分清楚。circRNA在调控心肌细胞发展、分化、增殖和凋亡中发挥重要作用，但在瓣膜钙化中的作用相关研究较少。我们发现circRNA_5303及下游靶基因Smad4表达在钙化的主动脉瓣膜中显著高表达，而miR-138-5p的表达明显降低，提示circRNA_5303可能在主动瓣膜钙化发生发展中起正向调控作用。本研究将通过对circRNA_5303-miR-138-5p-Smad4轴在hAVICs成骨样分化和钙化中的具体作用机制进行研究，并在基因工程小鼠模型上验证体外细胞水平的研究结果，从而阐明circRNA_5303在主动瓣膜钙化中的功能和具体调控机制，为临床这一类患者寻找干预治疗的分子靶点提供重要理论依据。

主动脉瓣钙化常见于钙磷代谢紊乱患者，是患者的主要心血管事件风险因素。已知microRNAs（miRNAs）在调节主动脉瓣钙化和成骨分化的细胞过程中发挥功能作用。然而，miRNA在钙磷代谢紊乱患者主动脉瓣成骨分化中的调节作用在很大程度上仍然未知。本研究旨在通过miRNA测序鉴定钙化人主动脉瓣间质细胞（human aortic valve interstitial cells, hAVICs）的差异性表达谱，探讨miR-93-5p和miR-374a-5p在hAVICs成骨分化中的功能作用和潜在机制。本研究在体外分离人hAVICs，用高钙/高磷酸盐培养基构建hAVICs体外钙化模型。通过miRNA高通量测序测定在非钙化hAVICs和钙化hAVICs中的miRNA表达谱，选择差异表达的miR-93-5p和miR-374a-5p进行后续研究。过表达或低表达miR-93-5p、miR-374a-5p后，使用茜素红染色、细胞内钙含量和碱性磷酸酶测定来评估hAVICs钙化和成骨分化。使用定量实时聚合酶链反应（RT-qPCR）和蛋白质印迹分析（WB）来评估Runt相关转录因子2（Runx2）的mRNA和蛋白质水平。通过生物信息学方法筛出相关靶基因，而后进行双荧光素酶基因报告分析、RT-qPCR和WB分析，以探讨骨形态发生蛋白2（BMP2）是否是miR-93-5p和miR-374a-5p的共同靶点。此外，以重组人BMP2（rhBMP2）进行拯救实验，以确定miR-93-5p和miR-374a-5p介导的高钙/高磷酸盐诱导的hAVICs成骨分化和钙化是否是通过BMP2/Smad1/5/RUNX2所致。结果表明，过表达miR-93-5p和miR-374a-5p有效地抑制了高钙/高磷酸盐诱导的细胞钙化和成骨分化。从机制上讲，miR-93-5p和miR-374a-5p进一步被揭示为通过靶向BMP2在高钙/高磷酸盐诱导的成骨分化中负调节p-Smad1/5和Runx2。在与miR-93-5p和/或miR-374a-5p模拟物结合rhBMP2孵育的hAVICs中，BMP2的低表达也被发现被逆转。综上，miR-93-5p和miR-374a-5p 通过调控BMP2/Smad1/5/Runx2 通路阻碍了与磷酸钙代谢失调相关的瓣膜间质细胞钙化和成骨分化。