中国组织工程研究 ›› 2023, Vol. 27 ›› Issue (19): 3023-3028.doi: 10.12307/2023.616

• 脂肪干细胞 adipose-derived stem cells • 上一篇    下一篇

miR-889-3p靶向Runx2抑制脂肪间充质干细胞的成骨分化

张耀天1,崔  军2,刘晶毅2   

  1. 1沈阳医学院,辽宁省沈阳市   110000;2沈阳医学院附属中心医院,辽宁省沈阳市   110000
  • 收稿日期:2022-06-08 接受日期:2022-07-28 出版日期:2023-07-08 发布日期:2022-11-28
  • 通讯作者: 崔军,硕士,沈阳医学院附属中心医院,辽宁省沈阳市 110000
  • 作者简介:张耀天,女,1998年生,四川省广汉市人,汉族,2016年沈阳医学院毕业,主要从事临床医学骨科相关研究。
  • 基金资助:
    辽宁省自然科学基金指导计划(20180550071),项目负责人:崔军

miR-889-3p inhibits osteogenic differentiation of adipose-derived mesenchymal stem cells by targeting Runx2

Zhang Yaotian1, Cui Jun2, Liu Jingyi2   

  1. 1Shenyang Medical College, Shenyang 110000, Liaoning Province, China; 2Affiliated Central Hospital of Shenyang Medical College, Shenyang 110000, Liaoning Province, China
  • Received:2022-06-08 Accepted:2022-07-28 Online:2023-07-08 Published:2022-11-28
  • Contact: Cui Jun, Master, Affiliated Central Hospital of Shenyang Medical College, Shenyang 110000, Liaoning Province, China
  • About author:Zhang Yaotian, Shenyang Medical College, Shenyang 110000, Liaoning Province, China
  • Supported by:
    Guidance Program of Liaoning Provincial Natural Science Foundation, No. 20180550071 (to CJ)

摘要:


文题释义:

MiRNAs:是长度为22个核苷酸的短RNA序列,通过与mRNA的3’-非翻译区互补结合负调控靶基因,调控多种细胞生物学进程,如增殖、分化、细胞生长、发育和死亡。一个miRNA可以靶向数百个mRNA并影响多种基因的表达。
Runx2:是Runx家族中的一员,它们具有共同的DNA结合域Runt,由Runx1、Runx2和Runx3组成,在骨骼发育中起到关键作用。由于Runx2 mRNA序列编码特异性蛋白质结构,Runx2被认为是骨形成和骨重塑的重要转录因子。

背景:研究表明miR-889-3p可参与调控多种细胞的增殖分化过程,但是其对脂肪间充质干细胞成骨分化的影响尚不明确。
目的:探讨miR-889-3p对脂肪间充质干细胞成骨分化的调节作用。
方法:体外分离、培养SD大鼠脂肪间充质干细胞,转染miR-889-3p模拟物(miR-889-3p mimic)、miR-889-3p抑制剂(miR-889-3p inhibitor)和阴性对照(miR-NC)并诱导成骨分化。RT-PCR和Western blot检测成骨诱导14 d后的碱性磷酸酶活性、Runx2、骨钙素、骨桥素、Osterix等成骨相关mRNA和蛋白的表达。将野生型Runx2、突变型Runx2分别与miR-889-3p模拟物和miR-NC共转染脂肪间充质干细胞,通过双荧光素酶报告基因检测miR-889-3p与Runx2的靶向关系。
结果与结论:①miR-889-3p表达水平随成骨诱导时间延长而逐渐减低;②成骨诱导第7天和第14天miR-889-3p inhibitor组的矿化结节数目、大小、颜色深浅明显超过miR-889-3p mimic组和miR-NC组(P < 0.05);③miR-889-3p 过表达后,碱性磷酸酶活性、Runx2、骨钙素、骨桥素、Osterix mRNA及蛋白表达量显著降低,而miR-889-3p敲除后成骨相关mRNA和蛋白表达量明显提高;④双荧光素酶报告基因检测结果显示miR-889-3p 过表达显著降低了野生型Runx2的荧光素酶活性;⑤上述结果表明,miR-889-3p通过靶向Runx2负向调控脂肪间充质干细胞成骨分化。

https://orcid.org/0000-0003-2118-581X (张耀天)

中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

关键词: miR-889-3p, Runx2, 脂肪间充质干细胞, 成骨分化, 骨缺损

Abstract: BACKGROUND: Studies have shown that miR-889-3p is able to participate in the regulation of the proliferation and differentiation of a variety of cells, but its effect on the osteogenic differentiation of adipose-derived mesenchymal stem cells remains unclear.  
OBJECTIVE: To investigate the regulation of miR-889-3p in osteogenic differentiation of adipose-derived mesenchymal stem cells.
METHODS: Adipose-derived mesenchymal stem cells from SD rats were isolated and cultured in vitro, transfected with miR-889-3p mimics, Mir-889-3p inhibitor and negative control (miR-NC) then induced osteogenic differentiation. RT-PCR and western blot assay were used to detect alkaline phosphatase activity, Runx2, osteocalcin, osteopontin, Osterix and other osteogenic related mRNA and protein expression levels. Adipose-derived mesenchymal stem cells were co-transfected with wild-type Runx2 and mutant Runx2 with miR-889-3p mimic and miR-NC, respectively. Luciferase reporter gene assay was used to detect the targeting relationship between miR-889-3p and Runx2.  
RESULTS AND CONCLUSION: (1) The expression level of miR-889-3p decreased gradually with the prolongation of osteogenic induction. (2) The number, size, and color depth of mineralized nodules in the miR-889-3p inhibitor group were significantly higher than those in the miR-889-3p mimic group and miR-NC group on days 7 and 14 of osteogenic induction (P < 0.05). (3) Overexpression of miR-889-3p significantly suppressed alkaline phosphatase activity, Runx2, osteocalcin, osteopontin, Osterix mRNA and protein expression, whereas the knockdown of miR-889-3p remarkably enhanced osteogenesis-related mRNA and protein expression. (4) The results of dual-luciferase reporter gene assay showed that overexpression of miR-889-3p significantly reduced the luciferase activity of wild-type Runx2. (5) These results suggest that miR-889-3p negatively regulates osteogenic differentiation of adipose-derived mesenchymal stem cells by targeting Runx2.

Key words: miR-889-3p, Runx2, adipose-derived mesenchymal stem cell, osteogenic differentiation, bone defect

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