Department of Hand and Foot Surgery, Brain Hospital of Hunan Province, No. 427, Section 3 of Furong Middle Road, Changsha, 410007, Hunan Province, China.
Department of Gastrointestinal Surgery, Second Xiangaya Hospital, Central South University, No. 139 Renmin Road, Furong District, Changsha, 410011, Hunan Province, China.
Mol Cell Biochem. 2022 Jan;477(1):283-293. doi: 10.1007/s11010-021-04276-1. Epub 2021 Oct 28.
Bone defect seriously affects the quality of life. Meanwhile, osteogenic differentiation in BMSCs could regulate the progression of bone defect. Transcription factors are known to regulate the osteogenic differentiation in BMSCs. The study aimed to investigate the detailed mechanism by which TP53 regulates the osteogenic differentiation. To study bone defect in vitro, BMSCs were isolated from spinal cord injury rats. CCK-8 assay was applied to test the cell viability. The mineralized nodules in BMSCs was tested by alizarin red staining. Meanwhile, TUNEL staining and flow cytometry were performed to test the cell apoptosis. mRNA expression was tested by qRT-PCR. Starbase and dual-luciferase reporter assay were used to predict the downstream mRNA of miR-2861. Moreover, western blot was applied to detect the protein expressions (TP53 and Smad7). BMSCs were successfully isolated from rats. The expressions of miR-2861 were significantly upregulated in osteogenic medium, compared with growth medium. MiR-2861 inhibitor significantly decreased the levels of OCN, ALP, BSP, and Runx2 in BMSCs. In addition, miR-2861 inhibitor notably inhibited the mineralized nodules, viability, and induced the apoptosis of BMSCs. Smad7 was identified to be the downstream target of miR-2861, and knockdown of Smad7 notably reversed miR-2861 inhibitor-induced inhibition of osteogenic differentiation and promotion of apoptosis in BMSCs. Moreover, miR-2861 was transcriptionally regulated by TP53 in BMSCs. TP53-meidiated miR-2861 promotes osteogenic differentiation of BMSCs by targeting Smad7. Thereby, our research might provide new methods for bone defect treatment.
骨缺损严重影响生活质量。同时，骨髓间充质干细胞（BMSCs）的成骨分化可以调节骨缺损的进展。转录因子被认为可以调节 BMSCs 的成骨分化。本研究旨在探讨 TP53 调节成骨分化的详细机制。为了体外研究骨缺损，从脊髓损伤大鼠中分离出 BMSCs。通过 CCK-8 assay 测试细胞活力。通过茜素红染色测试 BMSCs 中的矿化结节。同时，通过 TUNEL 染色和流式细胞术测试细胞凋亡。通过 qRT-PCR 测试 mRNA 表达。Starbase 和双荧光素酶报告基因 assay 用于预测 miR-2861 的下游 mRNA。此外，Western blot 用于检测蛋白表达（TP53 和 Smad7）。成功从大鼠中分离出 BMSCs。与生长培养基相比，成骨培养基中 miR-2861 的表达明显上调。miR-2861 抑制剂显著降低 BMSCs 中 OCN、ALP、BSP 和 Runx2 的水平。此外，miR-2861 抑制剂显著抑制矿化结节的形成、细胞活力，并诱导 BMSCs 凋亡。Smad7 被鉴定为 miR-2861 的下游靶标，Smad7 的敲低显著逆转了 miR-2861 抑制剂诱导的 BMSCs 成骨分化抑制和凋亡促进作用。此外，miR-2861 在 BMSCs 中受 TP53 的转录调控。TP53 介导的 miR-2861 通过靶向 Smad7 促进 BMSCs 的成骨分化。因此，我们的研究可能为骨缺损治疗提供新的方法。
