MicroRNA-23c 通过减少 FGF2 抑制间充质干细胞向软骨细胞分化，从而抑制关节软骨损伤的恢复。

MicroRNA-23c inhibits articular cartilage damage recovery by regulating MSCs differentiation to chondrocytes via reducing FGF2.

机构信息

Department of Orthopaedics, Changzhou Hospital of traditional Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Changzhou, China.

出版信息

Eur Rev Med Pharmacol Sci. 2019 Feb;23(3):941-948. doi: 10.26355/eurrev_201902_16980.

DOI:10.26355/eurrev_201902_16980

PMID:30779059

Abstract

OBJECTIVE

The aim of the study was to explore the role of microRNA-23c in the differentiation of marrow stromal cells (MSCs) to chondrocytes and its potential mechanism.

MATERIALS AND METHODS

MSCs were first isolated from rat bone marrow for cell culture. Surface antigens of MSCs (CD29 and CD34) were identified by flow cytometry. MSCs were induced for chondrogenic differentiation in MCDM (Mesenchymal Stem Cell Chondrogenic Differentiation Medium) for 0, 3, and 7 days, respectively, followed by detection of RUNX2, microRNA-23c and FGF2 expressions by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Alcian blue staining was performed to access proteoglycan deposition in MSCs transfected with microRNA-23c mimics or inhibitor. Western blot was conducted to detect the protein expressions of ACAN and COL2A1 in MSCs. The binding condition between microRNA-23c and FGF2 was verified by dual-luciferase reporter gene assay. Finally, MSCs were co-transfected with microRNA-23c mimics and FGF2 overexpression plasmid for rescue experiments.

RESULTS

On the fourth day of MSCs isolation, MSCs were in an elongated shape. Flow cytometry results showed positive expression of CD29 and negative expression of CD34, which were consistent with MSCs phenotype. QRT-PCR data elucidated that the mRNA levels of RUNX2 and FGF2 gradually increased, whereas microRNA-23c expression decreased with the prolongation of chondrogenic differentiation. Transfection of microRNA-23c mimics in MSCs remarkably elevated microRNA-23c expression. Alcian blue staining showed that microRNA-23c overexpression results in less proteoglycan deposition in MSCs than that of controls. Both mRNA and protein expressions of ACAN and COL2A1 decreased after microRNA-23c overexpression. Dual-luciferase reporter gene assay confirmed that FGF2 binds to microRNA-23c. Further Western blot results demonstrated that FGF2 expression is negatively regulated by microRNA-23c. FGF2 overexpression reversed the inhibitory effects of microRNA-23c on proteoglycan deposition, as well as expressions of ACAN and COL2A1.

CONCLUSIONS

MicroRNA-23c expression decreases during chondrogenic differentiation of MSCs, which inhibits MSCs differentiation to chondrocytes by inhibiting FGF2.

摘要

目的

本研究旨在探讨 microRNA-23c 在骨髓基质细胞（MSCs）向软骨细胞分化中的作用及其潜在机制。

材料和方法

首先从大鼠骨髓中分离出 MSCs 进行细胞培养。通过流式细胞术鉴定 MSCs 的表面抗原（CD29 和 CD34）。将 MSCs 分别在 MCDM（间充质干细胞软骨分化培养基）中诱导 0、3 和 7 天进行软骨分化，然后通过定量实时聚合酶链反应（qRT-PCR）检测 RUNX2、microRNA-23c 和 FGF2 的表达。用 Alcian 蓝染色检测转染 microRNA-23c 模拟物或抑制剂的 MSCs 中蛋白聚糖的沉积情况。通过 Western blot 检测 MSCs 中 ACAN 和 COL2A1 的蛋白表达。通过双荧光素酶报告基因检测验证 microRNA-23c 与 FGF2 的结合情况。最后，将 microRNA-23c 模拟物和 FGF2 过表达质粒共转染 MSCs 进行挽救实验。

结果

MSCs 分离的第 4 天，MSCs 呈长梭形。流式细胞术结果显示 CD29 阳性表达，CD34 阴性表达，符合 MSCs 表型。qRT-PCR 数据表明，随着软骨分化的延长，RUNX2 和 FGF2 的 mRNA 水平逐渐升高，而 microRNA-23c 的表达降低。转染 MSCs 的 microRNA-23c 模拟物可显著提高 microRNA-23c 的表达。Alcian 蓝染色显示，与对照组相比，microRNA-23c 过表达导致 MSCs 中蛋白聚糖沉积减少。microRNA-23c 过表达后，ACAN 和 COL2A1 的 mRNA 和蛋白表达均降低。双荧光素酶报告基因检测证实 FGF2 与 microRNA-23c 结合。进一步的 Western blot 结果表明，FGF2 的表达受 microRNA-23c 的负调控。FGF2 过表达逆转了 microRNA-23c 对蛋白聚糖沉积以及 ACAN 和 COL2A1 表达的抑制作用。