Yu Cheng, Wu Dannan, Zhao Chong, Wu Chaoguang
Department of Cardiac Surgery, Hainan General Hospital, No. 19, Xiuhua Road, Xiuying, Haikou, 570311, Hainan, China.
Department of Pharmacy, Hainan General Hospital, Haikou, 570311, Hainan, China.
J Bone Miner Metab. 2021 May;39(3):360-371. doi: 10.1007/s00774-020-01164-4. Epub 2020 Oct 18.
Calcified aortic valve disease (CAVD) is characterized by valve thickening and calcification. Osteoblast differentiation is one of the key steps of valve calcification. CircRNAs is involved in osteogenic differentiation of multiple mesenchymal cells. However, the function of circRNA TGFBR2 (TGFBR2) in CAVD remained unclear. We explored the effect and mechanism of TGFBR2 in modulating CAVD.
Human aortic valve interstitial cells (VICs) were subjected to osteogenic induction, and transfected with TGFBR2, miR-25-3p mimic and siTWIST1. The relationship between miR-25-3p and GFBR2 was predicted by starBase and confirmed by luciferase reporter and Person's correlation test. The relationship between miR-25-3p and TWIST1 was predicted by TargetScan and confirmed by luciferase reporter assay. The expressions of TGFBR2, miR-25-3p, TWIST1, osteoblast markers (RUNX2 and OPN) were detected by Western blot or/and qRT-PCR. Alkaline phosphatase (ALP) activity and calcium nodule was determined by colorimetric method and Alizarin Red S staining.
The expression of TGFBR2 was down-regulated and that of miR-25-3p was up-regulated in calcific valves and osteogenic VICs. TGFBR2 was inversely correlated with miR-25-3p expression in calcific valves. TGFBR2 sponged miR-25-3p to regulate TWIST1 expression in osteogenic VICs. During osteogenic differentiation, ALP activity, calcium nodule, the levels of osteoblast markers were increased in VICs. MiR-25-3p overexpression or TWIST1 knockdown reversed the inhibitory effect of TGFBR2 overexpression on ALP activity, calcium nodule, the expressions of RUNX2 and OPN in osteogenic VICs.
The findings indicated that TGFBR2/miR-25-3p/TWIST1 axis regulates osteoblast differentiation in VICs, supporting the fact that TGFBR2 is a miRNA sponge in CAVD.
钙化性主动脉瓣疾病（CAVD）的特征是瓣膜增厚和钙化。成骨细胞分化是瓣膜钙化的关键步骤之一。环状RNA（circRNA）参与多种间充质细胞的成骨分化。然而，circRNA TGFBR2在CAVD中的功能仍不清楚。我们探讨了TGFBR2在调节CAVD中的作用及机制。
对人主动脉瓣间质细胞（VICs）进行成骨诱导，并转染TGFBR2、miR-25-3p模拟物和siTWIST1。通过starBase预测miR-25-3p与TGFBR2之间的关系，并通过荧光素酶报告基因和皮尔逊相关性检验进行验证。通过TargetScan预测miR-25-3p与TWIST1之间的关系，并通过荧光素酶报告基因检测进行验证。通过蛋白质免疫印迹法或/和定量逆转录-聚合酶链反应（qRT-PCR）检测TGFBR2、miR-25-3p、TWIST1、成骨细胞标志物（RUNX2和骨桥蛋白（OPN））的表达。通过比色法和茜素红S染色测定碱性磷酸酶（ALP）活性和钙结节。
在钙化瓣膜和成骨VICs中，TGFBR2的表达下调，miR-25-3p的表达上调。在钙化瓣膜中，TGFBR2与miR-25-3p的表达呈负相关。TGFBR2通过海绵吸附miR-25-3p来调节成骨VICs中TWIST1的表达。在成骨分化过程中，VICs中的ALP活性、钙结节、成骨细胞标志物水平升高。miR-25-3p过表达或TWIST1敲低可逆转TGFBR2过表达对成骨VICs中ALP活性、钙结节、RUNX2和OPN表达的抑制作用。
研究结果表明，TGFBR2/miR-25-3p/TWIST1轴调节VICs中的成骨细胞分化，支持TGFBR2是CAVD中一种微小RNA海绵的事实。
