LncRNA FGD5-AS1调节miR-93-5p/BMP2轴促进人骨髓间充质干细胞成骨分化

doi:10.16352/j.issn.1001-6325.2023.08.1179

基础医学与临床 ›› 2023, Vol. 43 ›› Issue (8): 1179-1185.doi: 10.16352/j.issn.1001-6325.2023.08.1179

• 研究论文 • 下一篇

LncRNA FGD5-AS1调节miR-93-5p/BMP2轴促进人骨髓间充质干细胞成骨分化

张亮亮, 赵程锦, 周煜虎, 曹博, 段明明, 冯阳阳^*

延安大学附属医院创伤骨科,陕西延安716000

收稿日期:2022-05-09 修回日期:2022-12-09 出版日期:2023-08-05 发布日期:2023-07-26
通讯作者: ^*yyfeng2490@163.com
基金资助:
国家自然科学基金(82160431)

LncRNA FGD5-AS1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by regulating miR-93-5p/BMP2 axis

ZHANG Liangliang, ZHAO Chengjin, ZHOU Yuhu, CAO Bo, DUAN Mingming, FENG Yangyang^*

Department of Orthopedics and Trauma,Yan'an University Affiliated Hospital, Yan'an 716000, China

Received:2022-05-09 Revised:2022-12-09 Online:2023-08-05 Published:2023-07-26
Contact: ^*yyfeng2490@163.com

摘要/Abstract

摘要： 目的探讨长链非编码RNA(lncRNA)FGD5-AS1调节微小RNA miR-93-5p/骨形态发生蛋白-2(BMP2)轴对人骨髓间充质干细胞(hBM-MSCs)成骨分化的影响。方法取对数增殖期的hBM-MSCs,分别检测成骨分化前、后lncRNA FGD5-AS1、miR-93-5p、BMP2 mRNA表达水平;将细胞分别转染或共转染pcDNA FGD5-AS1、miR-93-5p inhibitor、miR-93-5p mimics及相应阴性对照,分组为pcDNA NC组、pcDNA FGD5-AS1组、inhibitor NC组、miR-93-5p inhibitor组、pcDNA FGD5-AS1+mimics NC组、pcDNA FGD5-AS1+miR-93-5p mimics组,取未转染细胞作为空白组;CCK-8法检测细胞增殖;碱性磷酸酶(ALP)试剂盒检测其活性;茜素红染色鉴定细胞矿化结节形成;Western blot检测BMP2及成骨相关标志物——骨钙素(OCN)、骨桥蛋白(OPN)、成骨相关转录因子(OSX)水平;双荧光素酶报告基因实验分别验证miR-93-5p与FGD5-AS1、BMP2的靶向关系。结果 miR-93-5p分别与FGD5-AS1、BMP2存在靶向关系。与分化前相比,hBM-MSCs成骨分化后lncRNA FGD5-AS1、BMP2 mRNA表达显著增加,miR-93-5p表达显著下降(P＜0.05);与pcDNA NC组相比,pcDNA FGD5-AS1组FGD5-AS1表达显著增加(P＜0.05),表明转染成功。后续实验发现矿化结节产生、细胞增殖、ALP活性及BMP2、OCN、OPN、OSX表达均显著增加(P＜0.05);与inhibitor NC组相比,miR-93-5p inhibitor组miR-93-5p表达降低,表明转染成功。后续实验发现矿化结节产生、细胞增殖、ALP活性及BMP2、OCN、OPN、OSX表达均显著增加(P＜0.05);miR-93-5p过表达可抑制FGD5-AS1对细胞成骨分化的促进作用(P＜0.05)。结论上调FGD5-AS1可以促进细胞成骨分化,可能与miR-93-5p/BMP2轴有关。

关键词: 人骨髓间充质干细胞, lncRNA FGD5-AS1, miR-93-5p/BMP2轴, 成骨分化

Abstract: Objective To investigate the impact of long non-coding RNA (lncRNA) FGD5-AS1 on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) through regulation of the microRNA miR-93-5p/bone morphogenetic protein-2(BMP2) axis. Methods hBM-MSCs in logarithmic growth phase were taken, and the expression levels of lncRNA FGD5-AS1, miR-93-5p and BMP2 mRNA were detected before and after osteogenic differentiation; The cells were transfected or co-transfected with pcDNA FGD5-AS1, miR-93-5p inhibitor, miR-93-5p mimics and corresponding negative controls, respectively, then divided into pcDNA NC group, pcDNA FGD5-AS1 group, inhibitor NC group, miR-93-5p inhibitor group, pcDNA FGD5-AS1+mimics NC group, or pcDNA FGD5-AS1+miR-93-5p mimics group and non-transfected cells were taken as blank group. CCK-8 assay was applied to detect cell proliferation ability of each group; Alkaline phosphatase (ALP) kit was applied to detect its activity; Alizarin red staining was applied to identify cellular mineralized nodule formation; Western blot was applied to detect the levels of BMP2, osteogenesis-related markers-osteocalcin (OCN), osteopontin (OPN), and osterix(OSX); Dual-luciferase experiment was applied to verify the targeting relationship of miR-93-5p with FGD5-AS1 and BMP2, respectively. Results lncRNA FGD5-AS1 and BMP2 were found to be targets of miR-93-5p. After osteogenic differentiation, the expression of FGD5-AS1 and BMP2 was increased and the expression of miR-93-5p was decreased (P＜0.05). Compared with pcDNA NC group, the expression of FGD5-AS1 in pcDNA FGD5-AS1 group was significantly increased(P＜0.05), indicating successful transfection; Mineralized nodules, cell proliferation, ALP activity and the expression of BMP2, OCN, OPN and OSX were obviously higher (P＜0.05) in pcDNA FGD5-AS1 group. Compared with the inhibitor NC group, the expression of miR-93-5p in miR-93-5p inhibitor group was significantly decreased (P＜0.05), indicating successful transfection; Mineralized nodules, cell proliferation, ALP activity and the expression of BMP2, OCN, OPN and OSX were obviously improved (P＜0.05) in miR-93-5p inhibitor group. Over-expression of miR-93-5p inhibited the promoting effect of FGD5-AS1 on the osteogenic differentiation of hBM-MSCs(P＜0.05). Conclusions Up-regulation of FGD-AS1 promotes the osteogenic differentiation of hBM-MSCs, which might be related to the miR-93-5p/BMP2 axis.

Key words: human bone marrow-derived mesenchymal stem cells, lncRNA FGD5-AS1, miR-93-5p/BMP2 axis, osteogenic differentiation

中图分类号:

R394.2

张亮亮, 赵程锦, 周煜虎, 曹博, 段明明, 冯阳阳. LncRNA FGD5-AS1调节miR-93-5p/BMP2轴促进人骨髓间充质干细胞成骨分化[J]. 基础医学与临床, 2023, 43(8): 1179-1185.

ZHANG Liangliang, ZHAO Chengjin, ZHOU Yuhu, CAO Bo, DUAN Mingming, FENG Yangyang. LncRNA FGD5-AS1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by regulating miR-93-5p/BMP2 axis[J]. Basic & Clinical Medicine, 2023, 43(8): 1179-1185.

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LncRNA FGD5-AS1调节miR-93-5p/BMP2轴促进人骨髓间充质干细胞成骨分化

LncRNA FGD5-AS1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells by regulating miR-93-5p/BMP2 axis

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