Liu Bin, Gan Wei, Jin Zhang, Wang Meng, Cui Guopeng, Zhang Hongyu, Wang Huafu
Department of Spine Surgery, The Sixth Affiliated Hospital of Wenzhou Medical University, Lishui People's Hospital, Lishui, China.
Pharmacy College, Wenzhou Medical University, Wenzhou, China.
Int J Stem Cells. 2021 Aug 30;14(3):286-297. doi: 10.15283/ijsc20188.
Osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) plays a critical role in the success of lumbar spinal fusion with autogenous bone graft. This study aims to explore the role and specific mechanism of miR-34c-5p in osteogenic differentiation of BMSCs.
Rabbit model of lumbar fusion was established by surgery. The osteogenic differentiation dataset of mesenchymal stem cells was obtained from the Gene Expression Omnibus (GEO) database, and differentially expressed miRNAs were analyzed using R language (limma package). The expressions of miR-34c-5p, miR-199a-5p, miR-324-5p, miR-361-5p, RUNX2, OCN and Bcl-2 were determined by qRT-PCR and Western blot. ELISA, Alizarin red staining and CCK-8 were used to detect the ALP content, calcium deposition and proliferation of BMSCs. The targeted binding sites between miR-34c-5p and Bcl-2 were predicted by the Target database and verified using dual-luciferase reporter assay. MiR-34c-5p expression was higher in rabbit lumbar fusion model and differentiated BMSCs than normal rabbit or BMSCs. The content of ALP and the deposition of calcium increased with the osteogenic differentiation of BMSCs. Upregulation of miR-34c-5p reduced cell proliferation and promoted ALP content, calcium deposition, RUNX2 and OCN expression compared with the control group. The effects of miR-34c-5p inhibitor were the opposite. In addition, miR-34c-5p negatively correlated with Bcl-2. Upregulation of Bcl-2 reversed the effects of miR-34c-5p on ALP content, calcium deposition, and the expressions of RUNX2 and OCN.
miR-34c-5p could promote osteogenic differentiation and suppress proliferation of BMSCs by inhibiting Bcl-2.
骨髓间充质干细胞（BMSCs）的成骨分化在自体骨移植腰椎融合术的成功中起着关键作用。本研究旨在探讨miR-34c-5p在BMSCs成骨分化中的作用及具体机制。
通过手术建立兔腰椎融合模型。从基因表达综合数据库（GEO）获取间充质干细胞的成骨分化数据集，使用R语言（limma包）分析差异表达的miRNA。采用qRT-PCR和蛋白质免疫印迹法检测miR-34c-5p、miR-199a-5p、miR-324-5p、miR-361-5p、RUNX2、OCN和Bcl-2的表达。采用酶联免疫吸附测定（ELISA）、茜素红染色和CCK-8检测BMSCs的碱性磷酸酶（ALP）含量、钙沉积和增殖情况。通过Target数据库预测miR-34c-5p与Bcl-2之间的靶向结合位点，并使用双荧光素酶报告基因检测法进行验证。在兔腰椎融合模型和分化的BMSCs中，miR-34c-5p的表达高于正常兔或BMSCs。随着BMSCs成骨分化，ALP含量和钙沉积增加。与对照组相比，miR-34c-5p上调降低细胞增殖并促进ALP含量、钙沉积、RUNX2和OCN表达。miR-34c-5p抑制剂的作用则相反。此外，miR-34c-5p与Bcl-2呈负相关。上调Bcl-2可逆转miR-34c-5p对ALP含量、钙沉积以及RUNX2和OCN表达的影响。
miR-34c-5p可通过抑制Bcl-2促进BMSCs的成骨分化并抑制其增殖。
