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2022, Vol. 26 ›› Issue (1): 70-75Mechanism by which miR-146a regulates osteogenic differentiation of adipose derived mesenchymal stem cellsHuang Tao1, Cheng Zhijian2, Jia Zhiqiang3, Zhao Xiaoguang1, Wang Lei1, Zhai Wenjing2, Zhou Yongxin1
Abstract: BACKGROUND: Previous studies have shown that miR-146a can affect the osteogenic differentiation of bone marrow mesenchymal stem cells, but its regulatory function in adipose derived mesenchymal stem cells has not been elucidated. OBJECTIVE: To investigate the regulatory role of miR-146a in the osteogenic differentiation of mouse adipose derived mesenchymal stem cells. METHODS: Adipose derived mesenchymal stem cells were obtained from male C57BL/6 mice. The cell morphology was observed by inverted microscope, and the expression of CD29, CD44 and CD45 of passage 3 cells was detected by flow cytometry. miR-NC, miR-146a mimics and miR-146a inhibitor were transfected into adipose derived mesenchymal stem cells for osteogenic differentiation. On day 6 of osteogenic induction, alkaline phosphatase staining was used to observe the degree of calcification. RT-PCR and western blot assay were used to detect the expression of osteogenic phenotype markers. On day 12 of osteogenic induction, alizarin red staining was used to identify the formation of mineralized matrix on the cell surface. RESULTS AND CONCLUSION: (1) The cultured cells were spindle shaped and fibroblast like. The expression of CD29 and CD44 was high and that of CD45 was low. (2) The expression of miR-146a in mouse adipose derived mesenchymal stem cells decreased with the prolongation of osteogenic induction time (P < 0.05). (3) Compared with miR-NC group, the amount and activity of alkaline phosphatase precipitates in miR-146a mimic group were significantly lower than those in miR-146a inhibitor group (P < 0.05). (4) Compared with miR-NC group, the mRNA and protein expressions of ALP, Runx2, Akt, p-Akt, PI3K and p-PI3K were significantly decreased in miR-146a mimic group, but significantly increased in miR-146a inhibitor group (P < 0.05). (5) Compared with miR-NC group, alizarin red staining showed that the degree of calcification in miR-146a mimic group was significantly lower (P < 0.05), while that in miR-146a inhibitor group was significantly higher (P < 0.05). (6) These results suggest that miR-146a can negatively regulate the differentiation of adipose derived mesenchymal stem cells into osteoblasts through PI3K/AKT signaling pathway. Key words:stem cells, adipose derived mesenchymal stem cells, osteoblasts, miR-146a, osteogenic differentiation, signaling pathway |