Liu Kai, Fan Xiao-E, Zhang Li, Yang Ying, Zhou Xiao-Ling
Department of Orthopedics, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an City, Shaanxi Province, China.
Department of Thoracic Surgery, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an City, Shaanxi Province, China.
Kaohsiung J Med Sci. 2022 Oct;38(10):992-1000. doi: 10.1002/kjm2.12564. Epub 2022 Jul 27.
Osteoarthritis (OA) is a degenerative joint disease, which is characterized by the degeneration of articular cartilage, thickening of subchondral bone, and inflammation of the synovial membrane. In this study, we aimed to investigate the effects and underlying mechanisms of circ-NCX1 in lipopolysaccharide (LPS)-induced injury in SW1353 chondrocytes, an in vitro model of OA. The levels of circ-NCX1, miR-133a, and related apoptotic proteins were determined by RT-qPCR. MTT assay was used to evaluate the cell viability. The apoptosis was determined by flow cytometry, whereas the expression of apoptosis proteins was detected by Western blot. Immunofluorescence was used to detect cleaved caspase-3 expression in cells. Luciferase reporter assay was used to verify the interaction between circ-NCX1 and miR-133a, and between miR-133a and Silent information regulator 2 homolog 1 (Sirt1). The results showed that the overexpression of circ-NCX1 significantly upregulated the chondrocyte viability and proliferation, and alleviated apoptosis in LPS-induced SW1353 cells. Immunofluorescence results showed that the overexpression of circ-NCX1 significantly reduced expression of LPS-stimulated cleaved Caspase-3. The RT-qPCR results showed that the overexpression of circ-NCX1 inhibited mRNA levels of cleaved Caspase-3 and Bax, and promoted mRNA levels of Bcl-2. Luciferase reporter assay showed that circ-NCX1 targeted miR-133a, and miR-133a directly targeted the Sirt1. In addition, overexpression of circ-NCX1 inhibited chondrocyte apoptosis and promoted Akt phosphorylation via the miR-133a/Sirt1 axis in LPS-induced chondrocytes. In conclusion, circ-NCX1 may serve as an important regulator of LPS-induced chondrocyte apoptosis through the miR-133a/Sirt1 axis, and may be involved in the development of OA.
骨关节炎（OA）是一种退行性关节疾病，其特征在于关节软骨退变、软骨下骨增厚以及滑膜炎症。在本研究中，我们旨在探讨环状钠钙交换体1（circ-NCX1）在脂多糖（LPS）诱导的SW1353软骨细胞损伤中的作用及潜在机制，SW1353软骨细胞是骨关节炎的一种体外模型。通过逆转录定量聚合酶链反应（RT-qPCR）测定circ-NCX1、微小RNA-133a（miR-133a）及相关凋亡蛋白的水平。采用噻唑蓝（MTT）比色法评估细胞活力。通过流式细胞术测定细胞凋亡，而凋亡蛋白的表达则通过蛋白质免疫印迹法检测。免疫荧光法用于检测细胞中裂解的半胱天冬酶-3（cleaved caspase-3）的表达。荧光素酶报告基因检测用于验证circ-NCX1与miR-133a之间以及miR-133a与沉默信息调节因子2同源物1（Sirt1）之间的相互作用。结果表明，circ-NCX1的过表达显著上调软骨细胞活力和增殖，并减轻LPS诱导的SW1353细胞凋亡。免疫荧光结果显示，circ-NCX1的过表达显著降低LPS刺激的裂解Caspase-3的表达。RT-qPCR结果显示，circ-NCX1的过表达抑制裂解Caspase-3和Bax的mRNA水平，并促进Bcl-2的mRNA水平。荧光素酶报告基因检测显示，circ-NCX1靶向miR-133a，且miR-133a直接靶向Sirt1。此外，circ-NCX1的过表达通过miR-133a/Sirt1轴抑制LPS诱导的软骨细胞凋亡并促进蛋白激酶B（Akt）磷酸化。总之，circ-NCX1可能通过miR-133a/Sirt1轴作为LPS诱导的软骨细胞凋亡的重要调节因子，并可能参与骨关节炎的发展。
