Department of Orthopedics, Changhai Hospital, Navy Medical University, Shanghai, People's Republic of China.
Connect Tissue Res. 2022 Sep;63(5):475-484. doi: 10.1080/03008207.2021.2019247. Epub 2022 Jan 12.
The etiology of adolescent idiopathic scoliosis (AIS) remains unclear. The chondrogenic differentiation of mesenchymal stem cells (MSCs) is important in AIS, and the Ras homolog gene family member A (RHOA) is associated with chondrogenesis. The purpose of this study was to explore the effect of RHOA on the chondrogenic differentiation of MSCs in AIS.
We isolated MSCs from patients with AIS (AIS MSCs) and individuals without AIS (control MSCs). The inhibitor Y27632 was used to inhibit the function of RHOA/ROCK signaling, and plasmid-based overexpression and siRNA-mediated knockdown were used to manipulate RHOA expression. CCK-8 was used to detect cell viability. The phosphorylation levels of LIMK1, MLC2 and cofilin were detected by Western blotting. The mRNA expression of aggrecan, SOX9, and COL2A1 were confirmed using RT-PCR. Immunofluorescence was used to analyze F-actin and collagen II. Alcian blue staining was performed to assess the secretion of glycosaminoglycans (GAGs).
We found that RHOA was significantly upregulated in AIS MSCs, and the phosphorylation levels of LIMK1, MLC2, and cofilin were increased. The mRNA expressions of aggrecan, SOX9, and COL2A1 were notably reduced in AIS MSCs. However, these effects were abolished by Y27632 treatment and RHOA knockdown in AIS MSCs. In addition, RHOA knockdown in AIS MSCs increased the content of collagen II and GAGs. RHOA overexpression in the control MSCs markedly activated the RHOA/ROCK signaling and decreased the expression of aggrecan, SOX9, and COL2A1, F-actin, and GAGs.
RHOA regulates the chondrogenic differentiation ability of MSCs in AIS via the RHOA/ROCK signaling pathway and this regulation may involve SOX9.
青少年特发性脊柱侧凸（AIS）的病因仍不清楚。间充质干细胞（MSCs）的软骨分化在 AIS 中很重要，Ras 同源基因家族成员 A（RHOA）与软骨生成有关。本研究旨在探讨 RHOA 对 AIS 中 MSCs 软骨分化的影响。
我们从 AIS 患者（AIS-MSCs）和无 AIS 个体（对照-MSCs）中分离 MSCs。使用 RHOA/ROCK 信号通路抑制剂 Y27632 抑制 RHOA 功能，使用质粒过表达和 siRNA 介导的敲低来操纵 RHOA 表达。CCK-8 法检测细胞活力。Western blot 检测 LIMK1、MLC2 和 cofilin 的磷酸化水平。RT-PCR 检测聚集蛋白聚糖、SOX9 和 COL2A1 的 mRNA 表达。免疫荧光法分析 F-肌动蛋白和胶原蛋白 II。阿尔辛蓝染色法评估糖胺聚糖（GAGs）的分泌。
我们发现 RHOA 在 AIS-MSCs 中显著上调，LIMK1、MLC2 和 cofilin 的磷酸化水平升高。AIS-MSCs 中聚集蛋白聚糖、SOX9 和 COL2A1 的 mRNA 表达明显降低。然而，这些作用在 AIS-MSCs 中被 Y27632 处理和 RHOA 敲低所消除。此外，AIS-MSCs 中 RHOA 敲低增加了胶原蛋白 II 和 GAGs 的含量。在对照 MSCs 中过表达 RHOA 显著激活了 RHOA/ROCK 信号通路，并降低了聚集蛋白聚糖、SOX9 和 COL2A1、F-肌动蛋白和 GAGs 的表达。
RHOA 通过 RHOA/ROCK 信号通路调节 AIS 中 MSCs 的软骨分化能力，这种调节可能涉及 SOX9。
