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- 2016
miR146通过靶向Notch 1促进骨髓间充质干细胞向髓核样细胞分化
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Abstract:
摘要：目的 探讨miR146在骨髓间充质干细胞(BMSC)向髓核样细胞分化中的作用及其机制。方法 从SD大鼠中分离培养BMSC，传至第3代用于本实验。以TGFβ1(转化生长因子-β1)诱导BMSC向髓核样细胞分化为模型。检测TGFβ1诱导BMSC后蛋白多糖(ACAN)、Ⅱ型胶原蛋白(COLⅡ)和SOX 9的蛋白及mRNA表达水平；RT-PCR检测miR146的表达水平。随后通过上调或下调miR146水平，检测ACAN、COLⅡ和SOX 9的蛋白及mRNA表达水平；双荧光素酶报告系统验证miR146的靶基因，并上调或下调miR146水平后检测靶基因的蛋白表达水平；过表达靶基因并检测其与miR146 mimic共转染对ACAN、COLⅡ和SOX 9表达的影响。最后，上调或下调靶基因后，检测miR146表达水平的变化。结果 TGFβ1诱导BMSC向髓核样细胞分化后ACAN、COLⅡ、SOX 9和miR146高表达。上调miR146后，ACAN、COLⅡ和SOX 9的表达升高；下调miR146后ACAN、COLⅡ和SOX 9的表达降低。双荧光素酶报告系统证实，Notch 1是miR146的靶基因，miR146通过抑制Notch 1的表达上调ACAN、COLⅡ和SOX 9的表达水平。上调Notch 1后miR146表达下调，下调Notch 1后miR146表达上调。结论 miR146通过靶向Notch 1促进骨髓间充质干细胞向髓核样细胞分化，Notch 1对miR146亦具有负向调控作用。
ABSTRACT: Objective To explore the effect of miR146 on the differentiation of bone mesenchymal stem cells (BMSCs) into nucleus pulposus-like cells and the underlying mechanism. Methods The BMSCs were isolated from SD mice. The 3 passages of BMSCs were used in the present study. The differentiation of BMSCs into pulposus-like cells was induced by TGFβ1. The protein and mRNA expression levels of aggrecan (ACAN), type Ⅱ collagen (COLⅡ) and SOX9 were detected in TGFβ1-induced BMSCs. The expression level of miR146 was determined by RT-PCR. By up-regulating or down-regulating the expression levels of miR146 in TGFβ1-induced BMSCs, we further detected the protein and mRNA expression levels of ACAN, COLⅡ and SOX9. The target gene of miR146 was verified by dual-luciferase reporter assay system. The miR146 was up-regulated or down-regulated in BMSCs and the protein expression levels of target gene were detected. The target gene was overexpressed and the expression levels of ACAN, COLⅡ and SOX9 were detected in BMSCs transfected by target gene and miR146 mimic. At last, the expression level of miR146 was detected by silencing or overexpressing the target gene. Results The expression levels of ACAN, COLⅡ, SOX 9 and miR146 were increased during the differentiation of BMSCs into pulposus-like cells induced by TGFβ1. After miR146 up-regulation, the expression levels of ACAN, COLⅡ and SOX 9 were elevated. After miR146 down-regulation, the expression levels of ACAN, COLⅡ and SOX 9 were reduced. Dual-luciferase reporter assay system showed that the target gene of miR146 was Notch 1. miR146 enhanced the expression levels of ACAN, COLⅡ and SOX 9 through inhibiting the expression of Notch 1. After up-regulating Notch 1, the expression of miR146 was reduced. After down-regulating Notch 1, the expression of miR146 was increased. Conclusion miR146 promotes the differentiation of BMSCs into nucleus pulposus-like cells by targeting
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