一氧化碳释放分子-3 通过 miR-195-5p/Wnt3a 通路增强大鼠骨髓间充质干细胞的成骨分化。
Carbon Monoxide-Releasing Molecule-3 Enhances Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells via miR-195-5p/Wnt3a Pathway.
机构信息
School and Hospital of Stomatology, Cheeloo College of Medicine, Shandong University & Shandong Key Laboratory of Oral Tissue Regeneration & Shandong Engineering Laboratory for Dental Materials and Oral Tissue Regeneration, Jinan, People's Republic of China.
Department of Oral and Maxillofacial Surgery, Shandong Linyi People's Hospital, Linyi, People's Republic of China.
出版信息
Drug Des Devel Ther. 2022 Jul 2;16:2101-2117. doi: 10.2147/DDDT.S367277. eCollection 2022.
PURPOSE
Bone marrow-derived mesenchymal stem cells (BMSCs) are hopeful in promoting bone regeneration as their pluripotency in differentiation. Our previous study showed that carbon monoxide-releasing molecule-3 (CORM-3) increased the osteogenic differentiation of rat BMSCs in vitro. However, the mechanism remained unclear. MicroRNAs (miRNAs) play a very important role in modulating the osteogenic differentiation of BMSCs. Therefore, we researched the miRNAs involved in CORM-3-stimulated osteogenic differentiation.
METHODS
The CORM-3-stimulated osteogenic differentiation of rat BMSCs was further studied in vivo. Based on the gene sequencing experiment, the rat BMSCs were transfected with miR-195-5p mimics and inhibitor, pcDNA3.1-Wnt3a and Wnt3a siRNA. The osteogenic differentiation of rat BMSCs was measured by quantitative real-time polymerase chain reaction, Western blot and alizarin red staining. Additionally, the targeting relationship between miR-195-5p and Wnt3a was confirmed by the dual-luciferase assay.
RESULTS
MiR-195-5p was down-expressed during the CORM-3-stimulated osteogenic differentiation of rat BMSCs. CORM-3-stimulated osteogenic differentiation of rat BMSCs was inhibited with miR-195-5p overexpression, evidenced by significantly reduced mRNA and protein expressions of runt-related transcription factor 2 and osteopontin, and matrix mineralization demonstrated. On the contrary, the osteogenic differentiation was enhanced with inhibition of miR-195-5p. CORM-3-stimulated osteogenic differentiation of rat BMSCs was increased by overexpression of Wnt3a, while the opposite was observed in the Wnt3a-deficient cells. Moreover, the decreased osteogenic differentiation capacity by increased expression of miR-195-5p was rescued by Wnt3a overexpression, showing miR-195-5p directly targeted Wnt3a.
CONCLUSION
These results demonstrate that CORM-3 promoted osteogenic differentiation of rat BMSCs via miR-195-5p/Wnt3a, which bodes well for the application of CORM-3 in the treatment of periodontal disease and other bone-defect diseases.
目的
骨髓间充质干细胞（BMSCs）具有多能性，在分化中具有潜力，有望促进骨再生。我们之前的研究表明，一氧化碳释放分子-3（CORM-3）可增加大鼠 BMSCs 的体外成骨分化。然而，其机制尚不清楚。microRNAs（miRNAs）在调节 BMSCs 的成骨分化中起着非常重要的作用。因此，我们研究了涉及 CORM-3 刺激成骨分化的 miRNAs。
方法
进一步研究了 CORM-3 刺激大鼠 BMSCs 的体内成骨分化。基于基因测序实验，用 miR-195-5p 模拟物和抑制剂、pcDNA3.1-Wnt3a 和 Wnt3a siRNA 转染大鼠 BMSCs。通过定量实时聚合酶链反应、Western blot 和茜素红染色来测量大鼠 BMSCs 的成骨分化。此外，通过双荧光素酶报告基因实验证实了 miR-195-5p 与 Wnt3a 之间的靶向关系。
结果
在 CORM-3 刺激大鼠 BMSCs 的成骨分化过程中，miR-195-5p 表达下调。miR-195-5p 过表达抑制 CORM-3 刺激的大鼠 BMSCs 成骨分化，表现为 runt 相关转录因子 2 和骨桥蛋白的 mRNA 和蛋白表达明显降低，基质矿化减少。相反，抑制 miR-195-5p 则增强了成骨分化。Wnt3a 过表达可增加 CORM-3 刺激的大鼠 BMSCs 的成骨分化，而在 Wnt3a 缺陷细胞中则观察到相反的结果。此外，过表达 miR-195-5p 降低的成骨分化能力可通过 Wnt3a 过表达得到挽救，表明 miR-195-5p 可直接靶向 Wnt3a。
结论
这些结果表明，CORM-3 通过 miR-195-5p/Wnt3a 促进大鼠 BMSCs 的成骨分化，这为 CORM-3 在治疗牙周病和其他骨缺损疾病中的应用提供了依据。
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