关键词: 骨髓间充质干细胞, 细胞衰老, miR-212-3p, 丝裂原活化蛋白激酶3, β-半乳糖苷酶

Abstract: BACKGROUND: Bone marrow mesenchymal stem cells in patients with osteoporosis show significant senescence and decreased activity and osteogenic differentiation. miR-212-3p inhibits osteogenic differentiation of human bone marrow mesenchymal stem cells. However, its regulation of senescence of bone marrow mesenchymal stem cells and its mechanism remain unclear.
OBJECTIVE: To investigate the effect of miR-212-3p on senescence of bone marrow mesenchymal stem cells by targeting mitogen-activated protein kinase 3 (MAPK3) and its mechanism. 
METHODS: Rat bone marrow mesenchymal stem cells were isolated and cultured in vitro, and the third generation was collected for the following experiments: (1) Cultured in two groups: The control group was added with complete culture medium, and the model group was added with complete culture medium containing H2O2. After 72 hours of culture, β-galactosidase activity, miR-212-3p and MAPK3 mRNA expression, as well as MAPK3, p16, and p21 protein expression were detected. (2) Cultured in three groups: control group, inhibitor control group, and miR-212-3p inhibitor group. After transfection for 24 hours, miR-212-3p, mRNA and protein expression of MAPK3 were detected. (3) Dual luciferase reporter gene combined with qRT-PCR and western blot assay were used to verify the targeting regulation of miR-212-3p and MAPK3. (4) Cultured in different groups: control inhibitor group, miR-212-3p inhibitor group, miR-212-3p inhibitor+interference control group, and miR-212-3p inhibitor+MAPK3 interference group. After transfection for 24 hours, MAPK protein and mRNA expression levels in cells were detected. They were divided into control group, H2O2 group, H2O2+ control inhibitor group, H2O2+miR-212-3p inhibitor group, H2O2+miR-212-3p inhibitor+interference control group, and H2O2+miR-212-3p inhibitor+MAPK3 interference group. Cells were transfected for 24 hours and then cultured with H2O2 for 72 hours. Aging-related β-galactosidase activity and p16 and p21 protein expression were detected.
RESULTS AND CONCLUSION: (1) Compared with the control group, β-galactosidase activity, miR-212-3p mRNA expression and p16, p21 protein expression were increased in the model group (P < 0.05), while MAPK3 mRNA and protein expression levels were decreased (P < 0.05). (2) Compared with the control group, the mRNA expression of miR-212-3p was decreased (P < 0.05), and the mRNA and protein expression levels of MAPK3 were increased (P < 0.05) in miR-212-3p inhibitor group. (3) Double luciferase reporter gene experiment confirmed that MAPK3 was the downstream target gene of miR-212-3p. (4) Compared with the control inhibitor group, the mRNA and protein expression levels of MAPK3 were increased in miR-212-3p inhibitor group (P < 0.05). Compared with the miR-212-3p inhibitor group, the mRNA and protein expression levels of MAPK3 in the miR-212-3p inhibitor+MAPK3 interference group were decreased (P < 0.05). Compared with H2O2+control inhibitor group, β-galactosidase activity in H2O2+miR-212-3p inhibitor group was decreased (P < 0.05). Compared with H2O2+miR-212-3p inhibitor group, β-galactosidase activity in H2O2+miR-212-3p inhibitor+MAPK3 interference group was higher than that in H2O2+miR-212-3p inhibitor group (P < 0.05). Compared with the H2O2+control inhibitor group, the protein expression levels of p16 and p21 in the H2O2+miR-212-3p inhibitor group were decreased (P < 0.05). Compared with H2O2+miR-212-3p inhibitor group, the protein expression levels of p16 and p21 in H2O2+miR-212-3p inhibitor+MAPK3 interference group were increased (P < 0.05). (5) To conclude, downregulation of miR-212-3p inhibits the senescence of rat bone marrow mesenchymal stem cells, and its mechanism of action may be achieved by targeting up-regulation of MAPK3 expression.

Key words: bone marrow mesenchymal stem cell, cell senescence, miR-212-3p, mitogen-activated protein kinase 3, β-galactosidase