Abstract：
BACKGROUND: Mesenchymal stem cells (MSCs) derived from the synovium, known as synovium mesenchymal stem cells (SMSCs), exhibit significant potential for articular cartilage regeneration owing to their capacity for chondrogenic differentiation. However, the microRNAs (miRNAs) governing this process and the associated mechanisms remain unclear. While mechanical stress positively influences chondrogenesis in MSCs, the miRNA-mediated response of SMSCs to mechanical stimuli is not well understood.
OBJECTIVE: This study explores the miRNA-driven mechano-transduction in SMSCs chondrogenesis under mechanical stress.
METHODS: The surface phenotype of SMSCs was analysed by flow cytometry. Chondrogenesis capacities of SMSCs were examined by Alcian blue staining. High throughput sequencing was used to screen mechano-sensitive miRNAs of SMSCs. The RNA expression level of COL2A1, ACAN, SOX9, BMPR2 and miR-143-3p of SMSCs were tested by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-143-3p and TLR4 was confirmed by luciferase reporter assays. The protein expression levels of related genes were assessed by western blot.
RESULTS: High-throughput sequencing revealed a notable reduction in miR-143-3p levels in mechanically stressed SMSCs. Gain- or loss-of-function strategies introduced by lentivirus demonstrated that miR-143-3p overexpression hindered chondrogenic differentiation, whereas its knockdown promoted this process. Bioinformatics scrutiny and luciferase reporter assays pinpointed a potential binding site for miR-143-3p within the 3\'-UTR of bone morphogenetic protein receptor type 2 (BMPR2). MiR-143-3p overexpression decreased BMPR2 expression and phosphorylated Smad1, 5 and 8 levels, while its inhibition activated BMPR2-Smad pathway.
CONCLUSIONS: This study elucidated that miR-143-3p negatively regulates SMSCs chondrogenic differentiation through the BMPR2-Smad pathway under mechanical tensile stress. The direct targeting of BMPR2 by miR-143-3p established a novel dimension to our understanding of mechano-transduction mechanism during SMSC chondrogenesis. This understanding is crucial for advancing strategies in articular cartilage regeneration.
OBJECTIVE: This study explores the miRNA-driven mechano-transduction in SMSCs chondrogenesis under mechanical stress.
METHODS: The surface phenotype of SMSCs was analysed by flow cytometry. Chondrogenesis capacities of SMSCs were examined by Alcian blue staining. High throughput sequencing was used to screen mechano-sensitive miRNAs of SMSCs. The RNA expression level of COL2A1, ACAN, SOX9, BMPR2 and miR-143-3p of SMSCs were tested by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-143-3p and TLR4 was confirmed by luciferase reporter assays. The protein expression levels of related genes were assessed by western blot.
RESULTS: High-throughput sequencing revealed a notable reduction in miR-143-3p levels in mechanically stressed SMSCs. Gain- or loss-of-function strategies introduced by lentivirus demonstrated that miR-143-3p overexpression hindered chondrogenic differentiation, whereas its knockdown promoted this process. Bioinformatics scrutiny and luciferase reporter assays pinpointed a potential binding site for miR-143-3p within the 3\'-UTR of bone morphogenetic protein receptor type 2 (BMPR2). MiR-143-3p overexpression decreased BMPR2 expression and phosphorylated Smad1, 5 and 8 levels, while its inhibition activated BMPR2-Smad pathway.
CONCLUSIONS: This study elucidated that miR-143-3p negatively regulates SMSCs chondrogenic differentiation through the BMPR2-Smad pathway under mechanical tensile stress. The direct targeting of BMPR2 by miR-143-3p established a novel dimension to our understanding of mechano-transduction mechanism during SMSC chondrogenesis. This understanding is crucial for advancing strategies in articular cartilage regeneration.
摘要：
背景：来自滑膜的间充质干细胞（MSCs），被称为滑膜间充质干细胞（SMSCs），由于它们的软骨分化能力，因此表现出关节软骨再生的显着潜力。然而,控制这一过程的microRNAs(miRNAs)及其相关机制尚不清楚.虽然机械应力对MSCs的软骨形成有积极的影响，miRNA介导的SMSCs对机械刺激的反应尚不清楚。
目的：本研究探讨了机械应力下SMSCs软骨形成中miRNA驱动的机械转导。
方法:通过流式细胞术分析SMSCs的表面表型。通过Alcian蓝染色检查SMSC的软骨形成能力。高通量测序用于筛选SMSCs的机械敏感性miRNA。COL2A1、ACAN、通过定量实时聚合酶链反应（qRT-PCR）检测SMSC的SOX9，BMPR2和miR-143-3p。miR-143-3p与TLR4之间的相互作用通过荧光素酶报告基因测定得到证实。蛋白质印迹法检测相关基因的蛋白表达水平。
结果：高通量测序显示，在机械应激的SMSCs中miR-143-3p水平显著降低。慢病毒引入的功能增益或丧失策略表明miR-143-3p过表达阻碍了软骨分化，而它的击倒促进了这一过程。生物信息学审查和荧光素酶报告基因测定在骨形态发生蛋白受体2型（BMPR2）的3'-UTR内确定了miR-143-3p的潜在结合位点。MiR-143-3p过表达降低了BMPR2表达和磷酸化Smad1、5和8水平，而其抑制作用激活了BMPR2-Smad通路。
结论：本研究阐明miR-143-3p在机械拉伸应力下通过BMPR2-Smad途径负调控SMSCs软骨分化。miR-143-3p对BMPR2的直接靶向为我们理解SMSC软骨形成过程中的机械转导机制建立了一个新的维度。这种理解对于推进关节软骨再生策略至关重要。
目的：本研究探讨了机械应力下SMSCs软骨形成中miRNA驱动的机械转导。
方法:通过流式细胞术分析SMSCs的表面表型。通过Alcian蓝染色检查SMSC的软骨形成能力。高通量测序用于筛选SMSCs的机械敏感性miRNA。COL2A1、ACAN、通过定量实时聚合酶链反应（qRT-PCR）检测SMSC的SOX9，BMPR2和miR-143-3p。miR-143-3p与TLR4之间的相互作用通过荧光素酶报告基因测定得到证实。蛋白质印迹法检测相关基因的蛋白表达水平。
结果：高通量测序显示，在机械应激的SMSCs中miR-143-3p水平显著降低。慢病毒引入的功能增益或丧失策略表明miR-143-3p过表达阻碍了软骨分化，而它的击倒促进了这一过程。生物信息学审查和荧光素酶报告基因测定在骨形态发生蛋白受体2型（BMPR2）的3'-UTR内确定了miR-143-3p的潜在结合位点。MiR-143-3p过表达降低了BMPR2表达和磷酸化Smad1、5和8水平，而其抑制作用激活了BMPR2-Smad通路。
结论：本研究阐明miR-143-3p在机械拉伸应力下通过BMPR2-Smad途径负调控SMSCs软骨分化。miR-143-3p对BMPR2的直接靶向为我们理解SMSC软骨形成过程中的机械转导机制建立了一个新的维度。这种理解对于推进关节软骨再生策略至关重要。
