miR-212-3p靶向MAPK3调控骨髓间充质干细胞的衰老  

miR-212-3p regulates senescence of bone marrow mesenchymal stem cells by targeting MAPK3

作　　者：钟丽颖 李顺东 王聪 Zhong Liying;Li Shundong;Wang Cong(Department of Geriatrics,Third Hospital of Changsha,Changsha 410015,Hunan Province,China)

机构地区：[1]长沙市第三医院老年病科,湖南省长沙市410015

出　　处：《中国组织工程研究》2025年第13期2690-2697,共8页Chinese Journal of Tissue Engineering Research

基　　金：长沙市自然科学基金(kq2208460),项目负责人:钟丽颖;湖南省自然科学基金(2021JJ70055),项目负责人:李顺东。

摘　　要：背景:骨质疏松症患者的骨髓间充质干细胞表现出明显衰老状态,并且细胞活性及成骨分化水平显著降低。miR-212-3p能够抑制人骨髓间充质干细胞的成骨分化,但其对骨髓间充质干细胞衰老调控的作用及机制尚不明确。目的:研究miR-212-3p通过靶向丝裂原活化蛋白激酶3(mitogen-activated protein kinase 3,MAPK3)对骨髓间充质干细胞衰老的影响及其机制。方法:体外分离培养大鼠骨髓间充质干细胞,收集第3代进行以下实验:①分2组培养:对照组加入完全培养基,造模组加入含H_(2)O_(2)的完全培养基培养,培养72 h后,检测细胞中β-半乳糖苷酶活性、miR-212-3p和MAPK3 mRNA表达,以及MAPK3、p16和p21蛋白表达。②分3组培养:对照组、抑制物对照组、miR-212-3p抑制物组,转染24 h后,检测细胞中miR-212-3p、MAPK3 mRNA表达及MAPK3蛋白表达。③采用双荧光素酶报告基因联合qRT-PCR和Western blot验证miR-212-3p与MAPK3靶向调控作用。④分组培养:分为对照抑制物组、miR-212-3p抑制物组、miR-212-3p抑制物+干扰对照组、miR-212-3p抑制物+MAPK3干扰组,转染24 h后,检测细胞中MAPK蛋白与mRNA表达。分为对照组、H_(2)O_(2)组、H_(2)O_(2)+对照抑制物组、H_(2)O_(2)+miR-212-3p抑制物组、H_(2)O_(2)+miR-212-3p抑制物+干扰对照组、H_(2)O_(2)+miR-212-3p抑制物+MAPK3干扰组,细胞转染24 h后再加入H_(2)O_(2)培养72 h,检测细胞中衰老相关β-半乳糖苷酶活性、p16和p21蛋白表达。结果与结论:①与对照组比较,造模组β-半乳糖苷酶活性、miR-212-3p mRNA表达及p16、p21蛋白表达升高(P<0.05),MAPK3 mRNA和蛋白表达降低(P<0.05)。②与对照组比较,miR-212-3p抑制物组细胞中miR-212-3p mRNA表达降低(P<0.05),MAPK3 mRNA与蛋白表达升高(P<0.05)。③双荧光素酶报告基因实验证实,MAPK3是miR-212-3p下游靶基因。④与对照抑制物组比较,miR-212-3p抑制物组细胞中MAPK3 mRNA和蛋白表达升高(P<0.05);与miR-212-3p抑制物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 H_(2)O_(2).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,H_(2)O_(2) group,H_(2)O_(2)+control inhibitor group,H_(2)O_(2)+miR-212-3p inhibitor group,H_(2)O_(2)+miR-212-3p inhibitor+interference control group,and H_(2)O_(2)+miR-212-3p inhibitor+MAPK3 interference group.Cells were transfected for 24 hours and then cultured with H_(2)O_(2) for 72 hours.Aging-relatedβ-galactosidase activity and p16 and p21 protein expression were detected.RESULTS AND CONCLU

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 

分 类 号：R459.9[医药卫生—治疗学] R363[医药卫生—临床医学] R681