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

doi:10.19438/j.cjoms.2018.04.003

中国口腔颌面外科杂志 ›› 2018, Vol. 16 ›› Issue (4): 302-309.doi: 10.19438/j.cjoms.2018.04.003

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

卢陈佩, 李洪亮, 欧阳宁鹃, 林雨恒, 司家文^*, 沈国芳^*

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

收稿日期:2018-01-10 修回日期:2018-03-13 出版日期:2018-07-20 发布日期:2018-08-09
通讯作者: 沈国芳,E-mail: maxillofacsurg@163.com;司家文,E-mail: sjwlyl@163.com。^*共同通信作者
作者简介:卢陈佩（1991-）,女,在读硕士研究生,E-mail: luchenpei@163.com
基金资助:
国家自然科学基金（81600827,81570947）

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

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; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology. Shanghai 200011, China;

Received:2018-01-10 Revised:2018-03-13 Online:2018-07-20 Published:2018-08-09

摘要/Abstract

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mRNA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。

关键词: microRNA-103-3p, 小鼠前成骨细胞MC3T3-E1, 成骨分化

Abstract: PURPOSE:To investigate the effect of miR-103-3p 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-103-3p 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-103-3p mimics and mimics negative control were separately transiently transfected to MC3T3-E1 cells with lipofectamine 2000. The expression of miR-103-3p 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 mRNA expression trends. miR-103-3p was suppressed during osteogenic differentiation of MC3T3-E1. After transient transfection of miR-103-3p mimics, the expression of miR-103-3p 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 for 3 and 7 days, the expression of Runx2, Osx and ALP on mRNA level was markedly down-regulated in the experimental group and so was the expression on protein level. CONCLUSIONS: miR-103-3p might suppress osteogenic differentiation of MC3T3-E1 cell at an early stage.

Key words: microRNA-103-3p, MC3T3-E1, Osteogenic differentiation

中图分类号:

Q254

卢陈佩, 李洪亮, 欧阳宁鹃, 林雨恒, 司家文, 沈国芳. MicroRNA-103-3p对小鼠前成骨细胞MC3T3-E1成骨分化早期的影响[J]. 中国口腔颌面外科杂志, 2018, 16(4): 302-309.

LU Chen-pei, LI Hong-liang, OUYANG Ning-juan, LIN Yu-heng, SI Jia-wen, SHEN Guo-fang. Impact of MicroRNA-103-3p on osteogenic differentiation of MC3T3-E1 at an early stage[J]. China J Oral Maxillofac Surg, 2018, 16(4): 302-309.

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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

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