hsa-circ-0107593 的下调通过 miR-20a-5p/SMAD6 信号促进 hADSCs 的成骨分化。

Down-regulation of hsa-circ-0107593 promotes osteogenic differentiation of hADSCs via miR-20a-5p/SMAD6 signaling.

机构信息

State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases & Other Research Platforms & Department of Orthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China.

State Key Laboratory of Oral Diseases & National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China.

出版信息

Oral Dis. 2023 Nov;29(8):3447-3459. doi: 10.1111/odi.14351. Epub 2022 Aug 30.

DOI:10.1111/odi.14351

PMID:35957556

Abstract

OBJECTIVES

Increasing evidence indicated circRNAs were involved in stem cells osteogenesis differentiation. Herein, we aimed to clarify the role of hsa-circ-0107593 during the osteogenesis process of human adipose-derived stem cells (hADSCs) and the underlying mechanisms.

METHODS

The ring structure of hsa-circ-0107593 was confirmed using RNase R treatment and Sanger sequencing. Nucleoplasmic separation and fluorescence in situ hybridization detected hsa-circ-0107593 distribution. Lentivirus and siRNA were used to modulate the expression of hsa-circ-0107593, and the binding relationship between hsa-circ-0107593 and miR-20a-5p was verified by luciferase assay and RNA immunoprecipitation. We detected the osteogenic activity of hADSCs through alkaline phosphatase staining, alizarin red S staining, real-time polymerase chain reaction (RT-PCR), western blot, and cellular immunofluorescence experiment. In vivo, micro-computed tomography was performed to analyze bone formation around skull defect.

RESULTS

RT-PCR results exhibited that hsa-circ-0107593 was downregulated while miR-20a-5p was upregulated during hADSCs osteogenesis. In vivo and in vitro experiments results indicated that knocking down hsa-circ-0107593 promoted the osteogenic differentiation of hADSCs, while overexpression of hsa-circ-0107593 showed an inhibitory effect on hADSCs osteogenic differentiation. In vitro experiment results showed hsa-circ-0107593 acted as a hADSCs osteogenic differentiation negative factor for it inhibited the suppressing effect of miR-20a-5p on SMAD6.

CONCLUSION

Knocking down hsa-circ-0107593 acts as a positive factor of the osteogenic differentiation of hADSCs via miR-20a-5p/SMAD6 signaling.

摘要

目的

越来越多的证据表明 circRNAs 参与干细胞成骨分化。在此，我们旨在阐明 hsa-circ-0107593 在人脂肪来源干细胞（hADSCs）成骨过程中的作用及其潜在机制。

方法

使用 RNase R 处理和 Sanger 测序证实 hsa-circ-0107593 的环状结构。核质分离和荧光原位杂交检测 hsa-circ-0107593 的分布。慢病毒和 siRNA 用于调节 hsa-circ-0107593 的表达，通过荧光素酶报告和 RNA 免疫沉淀验证 hsa-circ-0107593 与 miR-20a-5p 的结合关系。通过碱性磷酸酶染色、茜素红 S 染色、实时聚合酶链反应（RT-PCR）、western blot 和细胞免疫荧光实验检测 hADSCs 的成骨活性。体内实验，通过微计算机断层扫描分析颅骨缺损周围的骨形成。

结果

RT-PCR 结果显示，hADSCs 成骨过程中 hsa-circ-0107593 下调，miR-20a-5p 上调。体内和体外实验结果表明，敲低 hsa-circ-0107593 促进 hADSCs 的成骨分化，而过表达 hsa-circ-0107593 对 hADSCs 成骨分化有抑制作用。体外实验结果表明 hsa-circ-0107593 作为 hADSCs 成骨分化的负因子，因为它抑制了 miR-20a-5p 对 SMAD6 的抑制作用。