中国组织工程研究

中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (11): 1780-1786.doi: 10.12307/2023.129

• 骨组织构建 bone tissue construction • 上一篇    下一篇

miR-27b/PPARγ2轴对小鼠胚胎成骨前体细胞增殖和成骨细胞分化的意义

王  磊1，白雪松1，杜  宇1，何爱民1，郑  钧1，张志鹏1，吕惠成2   

  1. 1内蒙古医科大学，内蒙古自治区呼和浩特市  010010；2内蒙古医科大学第二附属医院创伤中心，内蒙古自治区呼和浩特市  010020
  • 收稿日期:2022-03-16 接受日期:2022-05-13 出版日期:2023-04-18 发布日期:2022-09-24
  • 通讯作者: 吕惠成，博士，教授，主任医师，内蒙古医科大学第二附属医院创伤中心，内蒙古自治区呼和浩特市 010020
  • 作者简介:王磊，男，1997年生，内蒙古自治区商都县人，汉族，内蒙古医科大学在读硕士，主要从事骨质疏松症方向的研究。 白雪松，男，1996年生，内蒙古自治区库伦旗库伦镇人，蒙古族，内蒙古医科大学在读硕士，主要从事骨质疏松症方向的研究。
  • 基金资助:
    内蒙古自治区自然科学基金(2017MS08118)，项目负责人：吕惠成；内蒙古医科大学科技百万专项(YKD2016kjbw010)，项目负责人：吕惠成

Significance of miR-27b/peroxisome proliferators-activated receptor gamma 2 axis for proliferation and osteoblast differentiation of mouse embryonic osteogenic precursor cells

Wang Lei1, Bai Xuesong1, Du Yu1, He Aimin1, Zheng Jun1, Zhang Zhipeng1, Lyu Huicheng2   

  1. 1Inner Mongolia Medical University, Hohhot 010010, Inner Mongolia Autonomous Region, China; 2Trauma Center, The Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010020, Inner Mongolia Autonomous Region, China
  • Received:2022-03-16 Accepted:2022-05-13 Online:2023-04-18 Published:2022-09-24
  • Contact: Lyu Huicheng, MD, Professor, Chief physician, Trauma Center, The Second Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010020, Inner Mongolia Autonomous Region, China
  • About author:Wang Lei, Master candidate, Inner Mongolia Medical University, Hohhot 010010, Inner Mongolia Autonomous Region, China Bai Xuesong, Master candidate, Inner Mongolia Medical University, Hohhot 010010, Inner Mongolia Autonomous Region, China Wang Lei and Bai Xuesong contributed equally to this work.
  • Supported by:
    the Natural Science Foundation of Inner Mongolia Autonomous Region, No. 2017MS08118 (to LHC); Inner Mongolia Medical University Science and Technology Million Project, No. YKD2016kjbw010 (to LHC)

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摘要：

文题释义：
骨质疏松症：一种代谢性骨病，其特征是骨密度和骨质量严重下降，骨微结构改变，导致骨骼变脆易于骨折。成骨细胞或破骨细胞的功能障碍可影响骨形成和吸收，最终导致骨质疏松症，在病理过程开始时常缺乏明显的症状，当出现骨折后，严重影响患者生活质量。
miRNA：是一类由内源基因编码的RNA分子，长度为20-22个核苷酸，可以通过结合靶mRNA的3’UTR区调控靶基因的表达抑制或翻译，从而在转录及转录后水平调控相关基因的表达。miRNA在细胞增殖、分化及凋亡等多种生物学活动过程中起着非常重要的调控作用。

背景：先前的研究证实了miR-27b在脂质水平的作用，可以控制多个对血脂异常有重要影响的基因，推测miR-27b可能通过靶向PPARγ2在骨质疏松症中发挥作用。
目的：探讨miR-27b/PPARγ轴在地塞米松诱导小鼠胚胎成骨细胞前体细胞MC3T3-E1细胞建立骨质疏松症细胞模型中的影响及相关作用机制。  
方法：体外地塞米松诱导MC3T3-E1细胞后，通过Lipofectamine®2000转染miR-27b模拟物、miR-27b抑制剂、NC-模拟物、NC-抑制剂、siPPARγ2、siNC，使用二甲基亚砜处理作为对照。细胞诱导培养后，检测细胞活性、碱性磷酸酶活性以确定细胞成骨、分化水平；实时荧光定量 PCR法和Western blot法检测成骨分化基因miR-27B、PPARγ2、Runx2、骨形态发生蛋白2和骨钙素的mRNA及蛋白表达水平；生信分析预测miR-27b下游靶基因，并用双荧光素酶基因报告实验验证。
结果与结论：①地塞米松处理显著降低了MC3T3-E1细胞的活力和miR-27b表达水平；与二甲基亚砜相比，地塞米松处理在24和48 h显著抑制MC3T3-E1细胞的活力并显著下调了miR-27b的表达水平；②miR-27b可直接调控PPARγ2；与相应的NC相比，miR-27b模拟物显著上调了miR-27b的表达水平，而miR-27b抑制剂显著下调了miR-27b的表达水平；与相应的NC相比，PPARγ2 mRNA和相应蛋白质表达被miR-27b模拟物抑制，并被miR-27b抑制剂显著增强；③miR-27b过表达减弱了地塞米松抑制MC3T3-E1细胞的增殖和成骨分化；与二甲基亚砜+ NC-mimic组相比，地塞米松显著抑制成骨细胞分化；而地塞米松介导的抑制作用则被miR-27b模拟物所逆转，细胞碱性磷酸酶活性和骨形态发生蛋白2、Runx2和骨钙素蛋白表达水平部分恢复；④抑制miR-27b通过上调PPARγ2抑制MC3T3-E1细胞的增殖和成骨分化；miR-27b的敲除明显增加了PPARγ2的表达，并降低了细胞活力、成骨细胞分化、碱性磷酸酶活性以及骨形态发生蛋白2、Runx2和骨钙素的表达水平；PPARγ2可能是miR-27b的直接作用靶点。

https://orcid.org/0000-0003-0629-4049(王磊)；https://orcid.org/0000-0003-2692-8053(白雪松)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: miR-27b, PPARγ2, MC3T3-E1, Runx2, 成骨分化, 增殖, 骨质疏松症, 地塞米松

Abstract: BACKGROUND: Previous studies have confirmed that miR-27b at the lipid level can control multiple genes that have an important impact on dyslipidemia. It is speculated that miR-27b may function in osteoporosis by targeting peroxisome proliferators-activated receptors γ2 (PPARγ2).
OBJECTIVE: To investigate the effect of miR-27b/PPARγ2 axis in the dexamethasone-induced osteoporosis model in mouse embryonic osteogenic precursor cells MC3T3-E1 and related mechanisms of action.
METHODS: After MC3T3-E1 cells were dexamethasone-induced and cultured in vitro, miR-27b mimic, miR-27b inhibitor, NC-mimic, NC-inhibitor, siPPARγ2, and siNC were transfected with Lipofectamine®2000, and dimethyl sulfoxide was used as a control. After the cells were induced and cultured, the cell viability and alkaline phosphatase activity were detected to determine the osteogenesis and differentiation levels of the cells. The mRNA and protein expression levels of osteogenic differentiation genes miR-27B, PPARγ2, Runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 and osteocalcin were detected by real-time fluorescence quantitative PCR and western blot methods. Bioinformatics analysis predicted the downstream target genes of miR-27b followed by verification with dual luciferase gene reporter experiments.
RESULTS AND CONCLUSION: Dexamethasone treatment significantly reduced cell viability and miR-27b expression levels in MC3T3-E1 pre-osteoblasts. Compared with dimethyl sulfoxide, dexamethasone significantly inhibited MC3T3-E1 cell viability and downregulated miR-27b expression levels at 24 and 48 hours. miR-27b could directly regulate PPARγ2. Compared with the corresponding NC-mimic, the miR-27b mimic significantly upregulated the expression level of miR-27b, while the miR-27b inhibitor significantly downregulated the expression level of miR-27b. Furthermore, PPARγ2 miRNA and protein expression levels were inhibited by the miR-27b mimic and significantly enhanced by the miR-27b inhibitor. miR-27b overexpression attenuated the inhibitory effect of dexamethasone on the proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts. Dexamethasone significantly inhibited osteoblast differentiation compared with the dimethyl sulfoxide + NC-mimic group. In contrast, the dexamethasone-mediated inhibition was reversed by the miR-27b mimic. Cellular alkaline phosphatase activity and bone morphogenetic protein 2, Runx2 and osteocalcin protein expression levels were partially restored. Inhibition of miR-27b suppressed proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts through the upregulation of PPARγ2. Knockout of miR-27b significantly increased PPARγ2 expression and decreased cell viability, osteoblast differentiation, alkaline phosphatase activity, and expression levels of bone morphogenetic protein 2, Runx2 and osteocalcin. PPARγ2 may be a direct target of miR-27b.

Key words: miR-27b, PPARγ2, MC3T3-E1, Runx2, osteogenic differentiation, proliferation, osteoporosis, dexamethasone

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