[1]丁永利,陈星,赵明明,等.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[J].中医正骨,2014,26(07):3-7.
　Ding Yongli*,Chen Xing,Zhao Mingming,et al.Effect of miR-133a on differentiation of murine preosteoblasts induced by bone morphogenetic protein 2 and the mechanism of action[J].The Journal of Traditional Chinese Orthopedics and Traumatology,2014,26(07):3-7.
点击复制

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()
分享到：

《中医正骨》[ISSN:1001-6015/CN:41-1162/R]

卷:
第26卷
期数:
2014年07期
页码:
3-7
栏目:
基础研究
出版日期:
2014-07-28

文章信息/Info

Title:
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; 陈星; 赵明明; 蔡一强; 姜勇
河南中医学院第一附属医院,河南 郑州 450000
Author(s):
Ding Yongli*;  Chen Xing; Zhao Mingming; Cai Yiqiang; Jiang Yong.
*The First Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450000,Henan,China
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
Keywords:
MicroRNAs;  Osteoblasts;  Bone morphogenetic protein 2;  miR-133a;  Runx-2;  Osterix;  Alkaline phosphatase;  Animal Experimentation
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RT-PCR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-2 mRNA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。
Abstract:
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 preosteoblast 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.

参考文献/References:

[1] Ambros V.The functions of animal microRNAs[J].Nature,2004,431(76):350-355.
[2] Ambros V.MicroRNA pathways in flies and worms:growth,death,fat,stress,and timing[J].Cell,2003,113(6):673-676.
[3] Lagos-Quintana M,Rauhut R,Lendeckel W,et al.Identification of novel genes coding for small expressed RNAs[J].Science,2001,294(5543):853-858.
[4] Lee RC,Ambros V.An extensive class of small RNAs in Caenorhabditis elegans[J].Science,2001,294(5543):862-864.
[5] Lee RC,Feinbaum RL,Ambros V.The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[J].Cell,1993,75(5):843-854.
[6] Liao XB,Zhang ZY,Yuan K,et al.miR-133a modulates osteogenic differentiation of vascular smooth muscle cells[J].Endocrinology,2013,154(9):3344-3352.
[7] Lee MH,Kim YJ,Kim HJ,et al.BMP-2-induced Runx2 expression is mediated by Dlx5,and TGF-beta 1 opposes the BMP-2-induced osteoblast differentiation by suppression of Dlx5 expression[J].J Biol Chem,2003,278(36):34387-34394.
[8] Lee MH,Kwon TG,Park HS,et al.BMP-2-induced Osterix expression is mediated by Dlx5 but is Independent of Runx2[J].Biochem Biophys Res Commun,2003,309(3):689-694.
[9] Hu Z,Peel SA,Ho SK,et al.Role of bovine bone morphogenetic proteins in bone matrix protein and osteoblast-related gene expression during rat bone marrow stromal cell differentiation[J].J Craniofac Surg,2005,16(6):1006-1014.
[10] Cai B,Pan Z,Lu Y.The roles of microRNAs in heart diseases:a novel important regulator[J].Curr Med Chem,2010,17(5):407-411.
[11] Chiyomaru T,Enokida H,Tatarano S,et al.miR-145 and miR-133a function as tumour suppressors and directly regulate FSCN1 expression in bladder cancer[J].Br J Cancer,2010,102(5):883-891.
[12] Small EM,Frost RJ,Olson EN.MicroRNAs add a new dimension to cardiovascular disease[J].Circulation,2010,121(8):1022-1032.
[13] Koutsoulidou A,Mastroyiannopoulos NP,Furling D,et al.Expression of miR-1,miR-133a,miR-133b and miR-206 increases during development of human skeletal muscle[J].BMC Dev Biol,2011,11:34.
[14] Li Z,Hassan MQ,Volinia S,et al.A microRNA signature for a BMP2-induced osteoblast lineage commitment program[J].Proc Natl Acad Sci U S A,2008,105(37):13906-13911.

更新日期/Last Update: 2014-07-28