miR-133a对骨形态发生蛋白2诱导的鼠前成骨细胞分化的影响及其作用机制  

Effect of miR-133a on differentiation of murine preosteoblasts induced by bone morphogenetic protein 2 and the mechanism of action

作　　者：丁永利[1] 陈星[1] 赵明明[1] 蔡一强[1] 姜勇[1] 

机构地区：[1]河南中医学院第一附属医院,河南郑州450000

出　　处：《中医正骨》2014年第7期3-7,共5页The Journal of Traditional Chinese Orthopedics and Traumatology

摘　　要：目的:探究miR-133a对骨形态发生蛋白2诱导的鼠前成骨细胞分化的影响及其作用机制。方法:检测骨形态发生蛋白2诱导的鼠前成骨细胞分化过程中miR-133a水平、碱性磷酸酶活性、Runx-2蛋白水平及Osterix蛋白水平,检测转染miR-133a后鼠前成骨细胞分化过程中碱性磷酸酶活性、Runx-2 mRNA水平、Runx-2蛋白水平、Osterix mRNA水平和Osterix蛋白水平。miR-133a测定采用qRT-PCR法,Runx-2和Osterix蛋白水平测定采用Western-blot法,Runx-2和Osterix mRNA水平测定采用RTPCR法,碱性磷酸酶活性检测采用碱性磷酸酶检测试剂盒。采用HEK293细胞进行miR-133a靶基因验证实验,细胞中荧光值的检测采用荧光素酶报告基因检测系统。结果:miR-133a在骨形态发生蛋白2诱导的MC3T3-E1细胞分化过程中表达显著下调,碱性磷酸酶的活性则显著增高。转染miR-133a后,细胞中碱性磷酸酶活性受到明显抑制,Runx-2蛋白水平显著下调、Runx-2mRNA水平未发生明显变化,Osterix的mRNA和蛋白水平均明显下调。荧光素酶报告基因检测结果显示,miR-133a可靶向作用于Runx-2 mRNA的3’-UTR区。结论:miR-133a通过抑制Runx-2的蛋白表达进而抑制骨形态发生蛋白2诱导的鼠前成骨细胞分化过程。Objective：To explore the effect of miR-133a on differentiation of murine preosteoblasts induced by bone morphogenetic protein 2（BMP-2）and the mechanism of action.Methods：The miR-133a levels,alkaline phosphatase（ALP）activities,Runx-2 protein levels and Osterix protein levels were detected in the process of murine preosteoblast differentiation induced by BMP 2.The ALP activities, Runx-2 mRNA levels,Runx-2 protein levels,Osterix mRNA levels and Osterix protein levels were detected in the process of murine preos-teoblast differentiation after miR-133a transfection.The qRT-PCR assays was used for miR-133a determination,Western-blot for Runx-2 and Osterix protein levels determination,RT-PCR for Runx-2 and Osterix mRNA levels determination and ALP Assay Kit for ALP activities determination.HEK293 cells were used to verify the target gene of miR-133a,and the luciferase reporter gene detection system was used for the determination of fluorescence value in cells.Results：A significant down-regulation of miR-133a expression was found in the process of MC3T3-E1 differentiation induced by BMP-2,while a significant increase was found in the ALP activities.After transfection of miR-133a, the ALP activities were inhibited significantly,and a significant down-regulation can be seen in the Runx-2 protein levels and the mRNA and protein levels of Osterix,while no evident changes was found in Runx-2 mRNA levels.The detection result of luciferase reporter gene showed that miR-133a can target the 3’-UTR region of Runx-2 mRNA.Conclusion：The miR-133a can suppress the murine preosteoblast differentiation induced by BMP-2 by suppressing the expression of Runx-2 protein.

关 键 词：微RNAS 成骨细胞 骨形态发生蛋白质2 miR-133a Runx-2 OSTERIX 碱性磷酸酶 动物实验 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]