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.
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。