高糖微环境下miR-449对人骨髓间充质干细胞成骨分化的作用和机制研究

Study on the Role and Mechanism of miR-449 on Osteogenic Differentiation of hBMSCs in High Glucose Microenvironment

目的探讨miR-449在高糖(HG)作用下对人骨髓间充质干细胞(hBMSCs)成骨分化的影响及其可能的作用机制。方法将hBMSCs接种于成骨培养基培养,诱导其成骨分化。实验组:成骨培养基中加入高糖25 mmol/L,模拟高糖微环境处理。对照组:细胞维持在5 mmol/L葡萄糖的正常培养基中。采用茜素红染色、碱性磷酸酶活性测定、双荧光素酶测定、实时定量PCR(qRT-PCR)检测等方法测定以下指标:(1)miR-449在HG诱导的hBMSCs成骨分化时的表达水平;(2)miR-449模拟物和抑制物对碱性磷酸酶(ALP)活性,成骨相关标志物表达的影响;(3)Sirt1和Fra-1的过表达对miR-449在HG处理的hBMSCs成骨分化标志物表达的影响。结果(1)HG处理的实验组hBMSCs成骨分化低于对照组细胞(P<0.05),miR-449表达水平较对照组降低(P<0.05)。(2)在HG诱导的hBMSCs成骨分化过程中,miR-449模拟物可降低ALP活性(P<0.05),降低成骨标志物的mRNA表达(P<0.05);而miR-449抑制剂能提高成骨标志物的mRNA活性(P<0.05)。(3)miR-449直接靶向结合Sirt1,Sirt1过表达逆转了miR-449模拟物对Fra-1mRNA表达的抑制作用,而Fra-1的过表达减轻了这种抑制作用;过表达Fra-1可减轻miR-449模拟物对ALP活性及成骨标志物mRNA的抑制作用。结论miR-449过表达通过Sirt1/Fra-1信号通路抑制HG处理的hBMSCs的成骨分化;通过调节miR-449可能为糖尿病骨质疏松症的干预提供一种新的治疗方法。

Objective To investigate the effect and possible mechanism of miR-449 on the osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs)with high glucose(HG).Methods hBMSCs were seeded in the osteogenic medium and induced to osteogenic differentiation.For the experimental group,osteogenic medium was supplemented with 25 mmol/L of high glucose to create a high glucose microenvironment.Control:cells were maintained in normal medium with 5 mmol/L of glucose.Alizarin red staining,alkaline phosphatase activity assay,dual luciferase assay,and real-time quantitative PCR(qRT-PCR)assay were used to determine the following indicators:①the expression levels of miR-449 in HG induced hBMSCs during osteogenic differentiation;②Effects of miR-449 mimic and inhibitor on alkaline phosphatase(ALP)activity,osteogenesis related markers expression;③Effects of overexpression of Sirt1 and Fra-1 on the expression of osteogenic differentiation markers by miR-449 in high glucose treated hBMSCs.Results①The osteogenic differentiation of hBMSCs in the experimental group with HG treatment was significantly lower than that of cells in the control group(P<0.05),and the miR-449 expression level increased(P<0.05);②During HG induced osteogenic differentiation of hBMSCs,miR-449 mimics significantly decreased ALP activity(P<0.05)and decreased the mRNA expression of osteogenic markers(P<0.05).While miR-449 inhibitor could increase ALP activity(P<0.05).③miR-449 directly targeted binding to SIRT1,and overexpression of Sirt1 reversed the inhibitory effect of miR-449 mimics on Fra-1 mRNA expression,whereas overexpression of Fra-1 alleviated this inhibitory effect.Overexpression of Fra-1 alleviated the inhibitory effect of miR-449 mimics on ALP activity and mRNA of osteogenic markers.Conclusion miR-449 overexpression inhibits osteogenic differentiation of HG-treated hBMSCs through the Sirt1/Fra-1 signaling pathway.Regulating miR-449 may provide a new therapeutic approach for the intervention of diabetic osteoporosis.