华西口腔医学杂志 ›› 2019, Vol. 37 ›› Issue (2): 200-207.doi: 10.7518/hxkq.2019.02.013

• 干细胞专栏 • 上一篇    下一篇

微小RNA-29a-3p调节卷曲蛋白4表达影响高脂血症大鼠种植体骨整合的实验研究

刘飞1,王志峰2,刘芳芳3,徐巾诏2,刘奇博1,蓝菁1()   

  1. 1.山东大学口腔医学院修复教研所,山东省口腔组织再生重点实验室
    2.山东大学口腔医院儿童牙病科,山东省口腔组织再生重点实验室,济南 250012
    3.南阳市口腔医院种植科,南阳 473000
  • 收稿日期:2018-10-30 修回日期:2019-01-05 出版日期:2019-04-01 发布日期:2019-04-28
  • 通讯作者: 蓝菁 E-mail:lanjing@sdu.edu.cn
  • 作者简介:刘飞,硕士,E-mail: xitieshiliufei@163.com
  • 基金资助:
    国家自然科学基金(81671025);山东省科技发展计划(2015GSF118186)

MicroRNA-29a-3p regulates osteoblast differentiation and peri-implant osseointegration in a rat model of hyperlipidemia by modulating Frizzled 4 expression

Fei Liu1,Zhifeng Wang2,Fangfang Liu3,Jinzhao Xu2,Qibo Liu1,Jing Lan1()   

  1. 1. Dept. of Prosthodontics, School of Stomatology, Shandong University, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan 250012, China
    2.Dept. of Pediatric Dentistry, School of Stomatology, Shandong University, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan 250012, China
    3.Dept. of Dental Implantology, Stomatological Hospital of Nanyang, Nanyang 473000, China
  • Received:2018-10-30 Revised:2019-01-05 Online:2019-04-01 Published:2019-04-28
  • Contact: Jing Lan E-mail:lanjing@sdu.edu.cn
  • Supported by:
    The National Natural Science Fundation of China(81671025);Shandong Science and Technology Development Plan(2015GSF118186)

摘要:

目的 研究microRNA-29a-3p(miR-29a-3p)对高脂环境下大鼠骨髓间充质干细胞(BMSCs)成骨分化和高脂血症大鼠种植体骨整合的影响及其作用位点。方法 1)体外实验:对BMSCs分别进行普通和高脂成骨诱导,通过逆转录实时定量聚合酶链反应和Western blot检测miR-29a-3p及成骨相关因子碱性磷酸酶(ALP)、Runt相关基因2(Runx2)的基因和蛋白质表达;高脂培养的BMSCs分别转染miR-29a-3p模拟物、抑制物及阴性对照(NC)质粒,RT-qPCR检测miR-29a-3p、ALP及Runx2基因表达情况,Western blot检测ALP、Runx2蛋白表达情况。通过靶基因预测软件(Target Scan、MiRNA.org等)预测miR-29a-3p与成骨相关的靶基因为卷曲蛋白4(Fzd4),双荧光素酶报告检测miR-29a-3p与Fzd4的相互作用关系。2)体内实验:高脂血症大鼠为实验组,普通大鼠为对照组,两组分别植入种植体,检测种植体周围骨组织中miR-29a-3p、ALP、Runx2的表达差异;行种植体-骨组织的硬组织切片,亚甲基蓝-酸性品红染色,进行组织学观察。分别将miR-29a-3p过表达慢病毒载体及空白对照慢病毒载体注射入高脂血症大鼠,种植体植入后3、10 d检测种植体周围骨组织中ALP、Runx2的表达变化以研究miR-29a-3p对高脂血症大鼠成骨的作用。结果 高脂组与普通组相比,ALP、Runx2和miR-29a-3p表达下调,BMSCs成骨分化能力下降。miR-29a-3p模拟物组与抑制物组相比,ALP、Runx2表达升高,BMSCs成骨分化能力增强。体内实验也得到了相似结果。双荧光素酶报告基因分析证实miR29a-3p通过直接结合3’-UTR抑制Fzd4表达。结论 miR-29a-3p对高脂环境下大鼠BMSCs成骨分化起正向调节作用,可直接与Fzd4结合调节成骨分化;miR-29a-3p能够促进高脂血症大鼠种植体周围成骨标志基因的表达,有利于骨整合。

关键词: 微小RNA-29a-3p, 高脂血症, 卷曲蛋白4, 双荧光素酶报告基因, 骨整合

Abstract:

Objective This work aimed to study and identify the influence and target gene of microRNA-29a-3p (miR-29a-3p) in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a high-fat environment in vitro and in vivo. Methods 1) In vitro: BMSCs were randomly allocated into two groups and were then induced to undergo osteogenic differentiation in a normal or high-fat environ-ment. Next, a miR-29a-3p mimic/inhibitor was transfected into the two groups of cells. The mRNA expression levels of alkaline phosphatase (ALP), Runt related gene 2 (Runx2), and miR-29a-3p and the protein expression levels of ALP and Runx2 were detected before and after transfection through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Moreover, Frizzled (Fzd) 4 was predicted as the target gene of miR-29a-3p by using an online database (Target Scan, MiRNA.org). The interactive relationship between miR-29a-3p and Fzd4 was confirmed through dual-luciferase assays. 2) In vivo: Rats were randomly divided into two groups and fed with a standard or high-fat diet. Titanium implants were grown in rats. Then, the expression levels of miR-29a-3p, ALP, and Runx2 were detected in bone tissues surrounding implants. Moreover, hard tissue sections were subjected to methylene blue-acid magenta staining and observed under microscopy to study bone formation around implants. In addition, miR-29a-3p-overexpressing lentiviral vectors were transfected into rats, and the expression levels of ALP, Runx2, and miR-29a-3p in bone tissues surrounding implants were detected at 3 and 10 days after transfection. Results The expression levels of ALP, Runx2, and miR-29a-3p and the osteogenic differentiation of BMSCs were suppressed in high-fat groups in vitro and in vivo. Conclusion MiR-29a-3p plays a positive role in the regulation of BMSCs in a high-fat environment. It can increase ALP and Runx2 expression levels in bone tissues surrounding implants in hyperlipidemia models. This result implies that miR-29a-3p can promote implant osseointergration in a rat model of hyperlipidemia.

Key words: microRNA-29a-3p, hyperlipidemia, Frizzled 4, dual luciferase reporter gene, osteointegration

中图分类号: 

  • R783

表1

基因及其引物序列"

引物名称 引物序列
ALP上游引物 5’-TGAGCGACACGGACAAGAAG-3’
ALP下游引物 5’-GCCTGGTAGTTGTTGTGAGCAT-3’
Runx2上游引物 5’-CACAAGTGCGGTGCAAACTT-3’
Runx2下游引物 5’-AATGACTCGGTTGGTCTCGG-3’
Fzd4上游引物 5’-GGAAGGACCAGGTGACGAAG-3’
Fzd4下游引物 5’-GGAATATGATGGGGCGCTCA-3’
mmu-miR-29a-3p 5’-UAACCGAUUUCAAAUGGUGCUA-3’
GAPDH上游引物 5’-TGATGGGTGTGAACCACGAG-3’
GAPDH下游引物 5’-CCCTTCCACGATGCCAAAGT-3’

图1

普通组和高脂组BMSCs成骨诱导28 d后检测 × 100 A:普通组,茜素红染色;B:高脂组,茜素红染色;C:普通组,油红O染色;D:高脂组,油红O染色。"

图2

普通组和高脂组BMSCs成骨诱导3、5、7、14 d的miR-29a-3p(左)、ALP(中)、Runx2(右)相对表达量"

图3

转染miR-29a-3p mimics/inhibitor 3 d后miR-29a-3p(左)、ALP(中)、Runx2(右)的相对表达量 A:mimics-NC组;B:miR-29a-3p mimics组;C:inhibitor-NC组;D:miR-29a-3p inhibitor组。"

图4

转染miR-29a-3p mimics/inhibitor 7 d后ALP和Runx2 mRNA及蛋白的表达 A:mimics-NC组;B:miR-29a-3p mimics组;C:inhibitor-NC组;D:miR-29a-3p inhibitor组。左:ALP表达;右:Runx2表达;上:mRNA表达水平检测;下:蛋白表达水平检测。"

表2

两组大鼠血清TC、TG、HDL、LDL检测结果"

血脂指标 对照组 实验组
TC 1.25±0.22 3.25±0.45*
TG 0.73±0.12 1.29±0.24*
HDL 0.91±0.16 1.21±0.95
LDL 0.29±0.06 0.65±0.12*

图5

野生型与突变型质粒与miR-29a-3p mimics/NC共转染后荧光素酶活性检测 A:NC+WT组;B:miR-29a-3p mimics+WT组;C:NC+Mut组;D:miR-29a-3p mimics+Mut组。"

图6

实验组与对照组种植体-骨结合情况 亚甲基蓝-酸性品红染色 A:对照组,× 100;B:实验组,× 100;C:对照组,× 200;D:实验组,× 200。"

图7

种植体植入5、10、15、20 d后miR-29a-3p(左)、ALP(中)和Runx2(右)的相对表达量"

图8

慢病毒转染3、10 d,高脂血症大鼠miR-29a-3p、ALP和Runx2表达变化 A:空白对照组;B:过表达组。上:过表达3 d,从左至右分别为miR-29a-3p、ALP和Runx2的mRNA相对表达量;下:过表达10 d,从左至右分别为:ALP mRNA相对表达量、Runx2 mRNA相对表达量、ALP和Runx2蛋白的相对表达量。"

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