MicroRNA-103-3p对小鼠前成骨细胞MC3T3-E1成骨分化早期的影响  

Impact of MicroRNA-103-3p on osteogenic differentiation of MC3T3-E1 at an early stage

作　　者：卢陈佩 李洪亮[1] 欧阳宁鹃 林雨恒[1] 司家文[1] 沈国芳[1] LU Chen-pei;LI Hong-liang;OUYANG Ning-juan;LIN Yu-heng;SI Jia-wen;SHEN Guo-fang(Department of Oral and Craniomaxillofacial Surgery,Shanghai Ninth People＇s Hospital,College of Stomatology,Shanghai Jiao Tong University School of Medicine t National Clinical Research Center for Oral Disease;Shanghai Key Laboratory of Stomatology ＆ Shanghai Research Institute of Stomatology.Shanghai 200011,China)

机构地区：[1]上海交通大学医学院附属第九人民医院.口腔医学院口腔颅颌面科国家口腔疾病临床研究中心上海市口腔医学重点实验室上海市口腔医学研究所,上海200011

出　　处：《中国口腔颌面外科杂志》2018年第4期302-309,共8页China Journal of Oral and Maxillofacial Surgery

基　　金：国家自然科学基金(81600827;81570947)

摘　　要：目的:探讨miR-103-3p对小鼠前成骨细胞MC3T3-E1成骨分化早期的影响。方法:以小鼠前成骨细胞MC3T3-E1为实验对象,对MC3T3-E1细胞进行成骨诱导,分别在0、3、5、7 d应用实时荧光定量PCR(real-time PCR)检测细胞中Runx2、Osx、ALP、miR-103-3p表达水平,Western免疫印迹(Western blotting)检测Runx2、Osx蛋白表达并进行碱性磷酸酶(ALP)染色。通过脂质体lipofectamine2000瞬时转染miR-103-3p模拟物(miR-103-3p mimics)及模拟物阴性对照进入MC3T3-E1细胞内,Real-time PCR检测2组细胞miR-103-3p的表达水平,CCK-8试剂盒检测细胞增殖。分别对2组细胞进行成骨诱导,在成骨诱导后0、3、7 d,分别使用Real-time PCR和Western免疫印迹检测2组细胞Runx2、Osx等成骨相关基因m RNA和蛋白的表达变化,并对2组细胞进行ALP染色。实验数据采用SPSS19.0软件包进行统计学分析。结果:MC3T3-E1经成骨诱导0、3、5、7 d后,细胞内Runx2、Osx、ALP转录水平持续显著升高;Runx2、Osx蛋白表达升高。ALP染色逐渐加深。在成骨诱导3、5、7 d的MC3T3-E1细胞中,miR-103-3p水平较诱导前受到持续显著抑制(P<0.05)。瞬时转染miR-103-3p mimics后,MC3T3-E1细胞中的miR-103-3p表达水平较对照组显著上调(P<0.05),细胞增殖受到抑制,Runx2、ALP转录水平显著抑制(P<0.05),Runx2蛋白表达显著抑制。对转染后的细胞进行成骨诱导3、7 d后,miR-103-3p转染组细胞Runx2、Osx、ALP在转录水平的表达较对照组显著降低,Runx2、Osx在蛋白水平的表达较对照组显著降低,且miR-103-3p转染组细胞ALP活性较对照组显著降低。结论:miR-103-3p可能对小鼠前成骨细胞MC3T3-E1的成骨分化早期起抑制作用。PURPOSE：To investigate the effect of miR-103-3 p on osteogenic differentiation of MC3T3-E1 cells at an early stage. METHODS： Osteogenic differentiation of MC3T3-E1 was induced in osteoblast induction medium. Runx2,Osx, ALP, miR-103-3 p mRNA expression was detected by real-time PCR at day 0, 3, 5 and 7 following induction;Western blotting was conducted to show Runx2, Osx protein expression. Osteoblastic phenotype was estimated by alkaline phosphatase（ALP） staining. miR-103-3 p mimics and mimics negative control were separately transiently transfected to MC3T3-E1 cells with lipofectamine 2000. The expression of miR-103-3 p was determined by real-time PCR. Cell proliferation was detected by CCK-8 kit. The transfected MC3T3-E1 cells were induced into osteogenic differentiation.Real-time PCR was conducted to detect the levels of osteogenesis-related genes, such as Runx2, Osx and ALP. Western blotting was performed to confirm their expression on protein level at day 0, 3 and 7 following induction. ALP activity was shown by ALP staining. The data were analyzed by SPSS 19.0 software package. RESULTS： mRNA expression levels of Runx2, Osx and ALP were increasingly up-regulated in MC3T3-E1 with osteogenic induction. Protein expression levels of Runx2 and Osx were consistent with m RNA expression trends. miR-103-3 p was suppressed during osteogenic differentiation of MC3T3-E1. After transient transfection of miR-103-3 p mimics, the expression of miR-103-3 p in the experimental group was significantly up-regulated（P〈0.05）. Cell proliferation was decreased. mRNA expression of Runx2 and ALP was markedly down-regulated（P〈0.05）. Runx2 protein expression was suppressed. After osteogenic induction for3 and 7 days, the expression of Runx2, Osx and ALP on m RNA level was markedly down-regulated in the experimental group and so was the expression on protein level. CONCLUSIONS： miR-103-3 p might suppress osteogenic differentiation of MC3T3-E1 cell at an early stage.

关 键 词：microRNA-103-3p 小鼠前成骨细胞MC3T3-E1 成骨分化 

分 类 号：Q254[生物学—细胞生物学]