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CircRNA TGFBR2/MiR-25-3p/TWIST1 axis regulates osteoblast differentiation of human aortic valve interstitial cells

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Abstract

Introduction

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.

Materials and Methods

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.

Results

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.

Conclusion

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.

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Authors and Affiliations

  1. Department of Cardiac Surgery, Hainan General Hospital, No. 19, Xiuhua Road, Xiuying, Haikou, 570311, Hainan, China

    Cheng Yu & Chaoguang Wu

  2. Department of Pharmacy, Hainan General Hospital, Haikou, 570311, Hainan, China

    Dannan Wu

  3. Department of English, School of Foreign Languages, Qiongtai Normal University, Haikou, 571127, Hainan, China

    Chong Zhao

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  1. Cheng Yu
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Contributions

Substantial contributions to conception and design: CY; data acquisition, data analysis and interpretation: DW, CZ, CW; drafting the article or critically revising it for important intellectual content: CY; final approval of the version to be published: all authors; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of the work are appropriately investigated and resolved: all authors.

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Correspondence to Cheng Yu.

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All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

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All patients had signed informed consent, and agreed that their tissues and cells would be used for any experimental work involving humans. The clinical trial program had been reviewed and approved by the Ethics Committee of Hainan General Hospital (Approval Number: HG20170305004).

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Yu, C., Wu, D., Zhao, C. et al. CircRNA TGFBR2/MiR-25-3p/TWIST1 axis regulates osteoblast differentiation of human aortic valve interstitial cells. J Bone Miner Metab 39, 360–371 (2021). https://doi.org/10.1007/s00774-020-01164-4

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