过表达miR-217调控EZH2促进骨质疏松模型小鼠BM-MSCs成骨分化

doi:10.16352/j.issn.1001-6325.2022.09.1406

基础医学与临床 ›› 2022, Vol. 42 ›› Issue (9): 1406-1413.doi: 10.16352/j.issn.1001-6325.2022.09.1406

过表达miR-217调控EZH2促进骨质疏松模型小鼠BM-MSCs成骨分化

阮锋¹, 李贺伟^1*, 龚艳琳², 刘佳丽¹, 刘平¹

1. 华中科技大学同济医学院附属梨园医院骨科,湖北武汉 430077;
2. 武汉市第一医院内分泌科,湖北武汉 430030

收稿日期:2022-01-10 修回日期:2022-06-10 出版日期:2022-09-05 发布日期:2022-09-02
通讯作者: lihewei198212@163.com
基金资助:
武汉市卫生健康委员会资助项目(武卫通【2019】87号)

Over-expression of miR-217 regulates EZH2 to promote osteogenic differentiation of BM-MSCs in osteoporotic mouse models

RUAN Feng¹, LI He-wei^1*, GONG Yan-lin², LIU Jia-li¹, LIU Ping¹

1. Department of Orthopedics, Liyuan Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430077;
2. Department of Endocrinology, the First Hospital of Wuhan, Wuhan 430030,China

Received:2022-01-10 Revised:2022-06-10 Online:2022-09-05 Published:2022-09-02

摘要/Abstract

摘要： 目的探究微小RNA-217(miR-217)对骨质疏松症(OP)模型小鼠骨髓间充质干细胞(BM-MSCs)成骨分化的影响及其可能调控机制。方法将小鼠分为OP组、假手术(sham)组,各12只,均进行了双侧卵巢切除术。将OP小鼠BM-MSCs分为对照组、miR-NC组、miR-217 mimics组、miR-217 mimics+pcDNA 3.1组、miR-217 mimics+pcDNA 3.1-EZH2(果蝇zeste基因增强子同源物2)组,分别不做转染、转染miR-NC、转染miR-217 mimics、转染miR-217 mimics及pcDNA 3.1、转染miR-217 mimics及pcDNA 3.1-EZH2,之后进行成骨诱导分化。RT-qPCR/Western blot检测miR-217及EZH2、OCN、Runx2、collagenⅠ mRNA和蛋白表达情况;酶标仪检测各组BM-MSCs碱性磷酸酶(ALP)活性;利用茜素红S(ARS)染色测定法对各组BM-MSCs进行染色,观察染色情况;双荧光素酶报告基因验证miR-217与EZH2的靶向关系。结果 OP组小鼠骨组织及BM-MSCs中miR-217表达水平低于sham组(P＜0.05),EZH2 mRNA表达水平高于sham组(P＜0.05)。miR-217 mimics组BM-MSCs中miR-217表达水平,OCN、Runx2、collagen Ⅰ mRNA和蛋白表达水平,ALP活性高于对照组和miR-NC组(P＜0.05),EZH2 mRNA及蛋白表达水平低于对照组和miR-NC组(P＜0.05),ARS染色程度重于对照组和miR-NC组。miR-217 mimics+pcDNA 3.1-EZH2组BM-MSCs中EZH2 mRNA及蛋白表达水平高于miR-217 mimics组、miR-217 mimics+pcDNA 3.1组(P＜0.05),OCN、Runx2、collagen Ⅰ mRNA和蛋白表达水平,ALP活性低于miR-217 mimics组、miR-217 mimics+pcDNA 3.1组(P＜0.05),ARS染色程度轻于miR-217 mimics组、miR-217 mimics+pcDNA 3.1组。miR-217与EZH2 mRNA 3′UTR区有结合位点,WT-EZH2+miR-217 mimics组荧光素酶相对活性低于WT-EZH2+miR-NC组(P＜0.05)。结论过表达miR-217可通过靶向抑制EZH2表达,促进OP小鼠BM-MSCs成骨分化,为临床防治OP提供新策略。

关键词: 微小RNA-217, 果蝇zeste基因增强子同源物2(EZH2), 骨髓间充质干细胞(BM-MSC), 骨质疏松症

Abstract: Objective To explore the effect of microRNA-217 (miR-217) on the osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs) in osteoporosis (OP) mouse model and potential regulatory mechanism. Methods The mice were divided into OP group and sham operation (sham) group with 12 in each group and both underwent bilateral oophorectomy. The OP mouse BM-MSCs were divided into control group, miR-NC group, miR-217mimics group, miR-217 mimics+pcDNA 3.1 group and miR-217 mimics+pcDNA 3.1-EZH2(enhancer of zeste homologe 2) group. The different groups were treated by transfection with miR-NC, miR-217 mimics, miR-217 mimics plus pcDNA 3.1, miR-217 mimics plus pcDNA 3.1-EZH2 respectively and then followed by osteogenic differentiation. The mRNA and protein expressions of miR-217, EZH2, OCN, Runx2, and collagen Ⅰ were detected by RT-qPCR/Western blot; The alkaline phosphatase (ALP) activity of BM-MSCs in each group was detected by microplate reader; Alizarin red S (ARS) staining assay was used to stain the BM-MSCs in each group for microscopy; The targeting relationship between miR-217 and EZH2 was verified by the dual luciferase reporter gene. Results The expression level of miR-217 in the bone tissue and BM-MSCs of the OP group was lower than that of the sham group (P＜0.05), and the expression level of EZH2 mRNA was higher than that of the sham group(P＜0.05). The expression level of miR-217 in BM-MSCs, the expression levels of OCN, Runx2, collagen Ⅰ mRNAs and proteins, and the ALP activity in the miR-217 mimics group were higher than those in the control group and in miR-NC group (P＜0.05). The expression level of EZH2 mRNA and protein were lower than those of the control group and miR-NC group (P＜0.05). And the degree of ARS staining was heavier than that of the control group and miR-NC group. The expression levels of EZH2 mRNA and protein in BM-MSCs in the miR-217 mimics+pcDNA 3.1-EZH2 group were higher than those in the miR-217 mimics group and miR-217 mimics+pcDNA 3.1 group (P＜0.05). And the expression levels of OCN, Runx2, collagen Ⅰ mRNAs and proteins, and ALP activity were lower than those in miR-217 mimics group and miR-217 mimics+pcDNA 3.1 group (P＜0.05). The degree of ARS staining was lighter than that in miR-217 mimics group and miR-217 mimics+pcDNA 3.1 group. There were binding sites between miR-217 and the 3′UTR region of EZH2 mRNA. The relative luciferase activity of WT-EZH2+miR-217 mimics group was lower than that of WT-EZH2+miR-NC group (P＜0.05). Conclusions Over-expression of miR-217 can inhibit the expression of EZH2 and promote the osteogenic differentiation of BM-MSCs in OP mice, which could provide a new strategy for clinical prevention and treatment of OP.

Key words: microRNA-217, enhancer of zeste homolog 2(EZH2), bone marrow derived mesenchymal stem cell(BM-MSC), osteoporosis

中图分类号:

R681

阮锋, 李贺伟, 龚艳琳, 刘佳丽, 刘平. 过表达miR-217调控EZH2促进骨质疏松模型小鼠BM-MSCs成骨分化[J]. 基础医学与临床, 2022, 42(9): 1406-1413.

RUAN Feng, LI He-wei, GONG Yan-lin, LIU Jia-li, LIU Ping. Over-expression of miR-217 regulates EZH2 to promote osteogenic differentiation of BM-MSCs in osteoporotic mouse models[J]. Basic & Clinical Medicine, 2022, 42(9): 1406-1413.

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过表达miR-217调控EZH2促进骨质疏松模型小鼠BM-MSCs成骨分化

Over-expression of miR-217 regulates EZH2 to promote osteogenic differentiation of BM-MSCs in osteoporotic mouse models

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