Li Yintai, Wei Suizhuan, Zhang Zhongping
Department of Rehabilitation, Baoji Traditional Chinese Medicine Hospital, Baoji, Shaanxi 721000, P.R. China.
Department of Orthopedics, Baoji Traditional Chinese Medicine Hospital, Baoji, Shaanxi 721000, P.R. China.
Exp Ther Med. 2021 Apr;21(4):407. doi: 10.3892/etm.2021.9838. Epub 2021 Feb 25.
Osteoarthritis (OA), characterized by the degeneration of articular cartilage, is a major problem in aging populations, and cartilage chondrocytes have been indicated to serve a curial role in the progression of OA. MicroRNA-200b-3p (miR-200b) was preliminarily identified to participate in OA. However, its role and mechanism of action in injured chondrocytes in OA remain unclear to date. In the present study, lipopolysaccharide (LPS)-treated cells isolated from normal knee articular cartilage were used to mimic inflammatory injury of OA chondrocytes. Cell viability, apoptosis and inflammatory responses were detected using Cell Counting Kit-8, flow cytometry and enzyme-linked immunosorbent assay, respectively. The expression levels of miR-200b and fucosyltransferase-4 (FUT4) were measured by reverse transcription-quantitative PCR and western blotting. The association between miR-200b and FUT4 was verified using TargetScan software, dual-luciferase reporter assay and RNA immunoprecipitation. The results indicated that LPS treatment decreased cell viability of primary chondrocytes, and increased apoptosis rate and production of IL-1β, IL-6 and TNF-α. The expression level of miR-200b was downregulated, and that of FUT4 was upregulated in OA cartilage tissues and LPS-treated normal chondrocytes compared with normal cartilage tissues and chondrocytes. Overexpression of miR-200b via transfection with miR-200b mimic inhibited the apoptosis rate and reduced the levels of IL-1β, IL-6 and TNF-α in LPS-stimulated chondrocytes. However, the suppressive effect of miR-200b overexpression on the LPS-induced inflammatory injury in chondrocytes was reversed by the restoration of FUT4 levels. Notably, FUT4 was indicated to be a downstream target of miR-200b and was negatively regulated by miR-200b. Taken together, the results of the current study indicated that miR-200b protected chondrocytes from LPS-induced inflammatory injury by targeting FUT4. These findings revealed the miR-200b/FUT4 axis as a potential candidate to target the degeneration of cartilages, thereby inhibiting the progression of OA.
骨关节炎（OA）以关节软骨退变为特征，是老龄人群中的一个主要问题，并且软骨细胞已被表明在OA进展中起关键作用。微小RNA-200b-3p（miR-200b）被初步鉴定参与OA。然而，其在OA损伤软骨细胞中的作用及作用机制迄今仍不清楚。在本研究中，使用从正常膝关节软骨分离的经脂多糖（LPS）处理的细胞来模拟OA软骨细胞的炎性损伤。分别使用细胞计数试剂盒-8、流式细胞术和酶联免疫吸附测定法检测细胞活力、凋亡和炎性反应。通过逆转录定量PCR和蛋白质印迹法测量miR-200b和岩藻糖基转移酶-4（FUT4）的表达水平。使用TargetScan软件、双荧光素酶报告基因测定法和RNA免疫沉淀法验证miR-200b与FUT4之间的关联。结果表明，LPS处理降低了原代软骨细胞的细胞活力，并增加了凋亡率以及白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的产生。与正常软骨组织和软骨细胞相比，OA软骨组织和经LPS处理的正常软骨细胞中miR-200b的表达水平下调，而FUT4的表达水平上调。通过转染miR-200b模拟物过表达miR-200b可抑制LPS刺激的软骨细胞的凋亡率，并降低IL-1β、IL-6和TNF-α的水平。然而，FUT4水平的恢复逆转了miR-200b过表达对软骨细胞中LPS诱导的炎性损伤的抑制作用。值得注意的是，FUT4被表明是miR-200b的下游靶点，并受到miR-200b的负调控。综上所述，本研究结果表明，miR-200b通过靶向FUT4保护软骨细胞免受LPS诱导的炎性损伤。这些发现揭示了miR-200b/FUT4轴作为靶向软骨退变从而抑制OA进展的潜在候选靶点。
