miR-155通过抑制SMAD5调控成骨细胞骨分化

发布时间：2018-07-29 17:17

【摘要】：背景:诱导成骨细胞分化为骨细胞是组织工程中的研究热点,但分化过程中的调节机制尚未完全阐明。MicroR NA是一类内源性小RNA分子,可通过与靶基因mR NA的3’端非编码区结合在转录后水平调控基因表达。研究证实,microR NA在骨细胞分化过程中起到重要的调节作用。目的:研究mi R-155对成骨细胞骨分化的调节作用以及作用机制。方法:取小鼠成骨细胞系MC3T3-E1细胞,加入小鼠骨形态发生蛋白2(200μg/L)进行成骨诱导,并与诱导第1,3,7,14天,通过qR T-PCR检测mi R-155 m RNA的表达。并将MC3T3-E1细胞分为对照组、诱导组、miR-155组和mi R-155 inhibitor组,对照组用α-MEM培养基培养,诱导组在培养基中加入骨形态发生蛋白2进行成骨诱导,mi R-155组和miR-155 inhibitor组在成骨诱导之前分别转染miR-155和mi R-155拮抗剂,诱导2周。结果与结论:(1)茜素红染色及碱性磷酸酶活性测定:经骨形态发生蛋白2诱导后,mi R-155 mR NA表达呈时间依赖性下调。诱导组MC3T3-E1细胞茜素红着色强度、碱性磷酸酶活性和mR NA表达也显著高于对照组(P0.01);mi R-155组茜素红着色强度明显低于诱导组,碱性磷酸酶活性和mR NA也明显低于对照组(P0.01),而mi R-155 inhibitor组则呈反向变化(P0.05);(2)荧光报告基因检测和western blot检测:miR-155可与SMAD5 mR NA 3’非编码区结合,并明显降低MC3T3-E1细胞中SMAD5蛋白和mR NA表达(P0.01)。结果表明,mi R-155可通过负性调控SMAD5表达,抑制MC3T3-E1细胞的成骨分化。
[Abstract]:Background: osteoblast differentiation into osteoblasts is a hot topic in tissue engineering. However, the regulatory mechanism of osteoblast differentiation has not been fully elucidated. MicroRNA is a class of endogenous small RNA molecules. Gene expression can be regulated at post-transcriptional level by binding to the 3 '-terminal noncoding region of target gene mRNA. MicroRNA plays an important role in the process of osteocyte differentiation. Aim: to study the effect and mechanism of mi R 155 on bone differentiation of osteoblasts. Methods: bone morphogenetic protein 2 (200 渭 g / L) was added to mouse osteoblast cell line MC3T3-E1 to induce osteogenesis. The expression of mi R-155 m RNA was detected by QR T-PCR on the 7th and 14th day after induction. MC3T3-E1 cells were divided into control group, induction group and miR-155 inhibitor group. The control group was cultured on 伪 -MEM medium. In the induction group, bone morphogenetic protein 2 was added to the culture medium to induce osteogenesis. The miR-155 and mi R-155 antagonists were transfected into the culture medium for 2 weeks. Results and conclusion: (1) alizarin red staining and alkaline phosphatase activity: the expression of mi R-155mR na was down-regulated in a time-dependent manner after induction by bone morphogenetic protein 2 (BMP 2). The intensity of alizarin red staining, alkaline phosphatase activity and mRNA expression in MC3T3-E1 cells in induction group were significantly higher than those in control group (P0.01). The activity of alkaline phosphatase and mRNA were also significantly lower than those of control group (P0.01), while the expression of SMAD5 protein and mRNA in MC3T3-E1 cells decreased significantly (P0.01), while the expression of SMAD5 protein and mRNA in MC3T3-E1 cells were significantly decreased by fluorescence reporter gene detection (P0.05); (2) and western blot detection (P0.01). The results showed that R- 155 could inhibit the osteogenic differentiation of MC3T3-E1 cells through negative regulation of SMAD5 expression.
【作者单位】：中国医科大学附属盛京医院南湖院区创伤骨科;朝阳市中心医院神经内科;吉林大学第一医院干部病房;
【基金】：国家自然科学基金(81600016)~~
【分类号】：R318.08;R68

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