Department of Orthopaedics (Trauma Orthopaedics Ward), The First Affiliated Hospital of Anhui Medical University, Hefei, Anhui 230022, P.R. China.
Mol Med Rep. 2019 Sep;20(3):2633-2640. doi: 10.3892/mmr.2019.10493. Epub 2019 Jul 12.
Osteoarthritis (OA) is a type of degenerative joint disease that affects the health of the elderly. OA is characterized by articular cartilage degradation and joint inflammation. The present study aimed to investigate the role and mechanism of microRNA‑let‑7a (Let‑7a) in OA by examining its role in lipopolysaccharide (LPS)‑induced cartilage inflammatory injury in ATDC5 cells. ATDC5 cells were treated with various concentrations of LPS. The present results suggested that 5 and 10 µg/ml LPS significantly inhibited ATDC5 cell viability, and 5 µg/ml LPS was selected for further experiments. Reverse transcription‑quantitative PCR (RT‑qPCR) results suggested that treatment with LPS significantly induced the expression levels of multiple inflammatory factors, including tumor necrosis factor‑α (TNF‑α), interleukin (IL)‑1β, IL‑6 and IL‑8, and increased the expression level of Let‑7a in ATDC5 cells. IL‑6 receptor (IL‑6R) was identified to be a direct target of Let‑7a using TargetScan and a dual‑luciferase reporter assay. Subsequently, Cell Counting Kit‑8 and flow cytometry analyses identified that Let‑7a inhibitor could significantly promote cell viability and reduce cell apoptosis in ATDC5 cells treated with LPS, and these effects could be reversed by transfection with small interfering (si)RNA‑IL‑6R. ELISA was used to examine the expression of inflammatory factors in ATDC5 cells following treatment with LPS. Additionally, RT‑qPCR and western blotting were performed to detect the mRNA and protein expression level of IL‑6R and STAT3. The present results suggested that Let‑7a inhibitor significantly reduced the expression level of TNF‑α, IL‑1β, IL‑6 and IL‑8 in ATDC5 cells, and this effect was reversed by transfecting siRNA‑IL‑6R. Moreover, RT‑qPCR and western blot assay results suggested that Let‑7a inhibitor significantly increased the expression level of IL‑6R and phosphorylated STAT3, and these effects could be reversed by siRNA‑IL‑6R. Collectively, Let‑7a inhibitor increased cell proliferation, reduced apoptosis and inhibited inflammatory response in ATDC5 cells treated with LPS. The present study provided a new potential therapeutic target for OA treatment.
骨关节炎（OA）是一种影响老年人健康的退行性关节疾病。OA 的特征是关节软骨降解和关节炎症。本研究旨在通过研究其在脂多糖（LPS）诱导的 ATDC5 细胞软骨炎症损伤中的作用，探讨 microRNA-let-7a（Let-7a）在 OA 中的作用。用不同浓度的 LPS 处理 ATDC5 细胞。结果表明，5 和 10μg/ml LPS 显著抑制 ATDC5 细胞活力，选择 5μg/ml LPS 进行进一步实验。逆转录-定量 PCR（RT-qPCR）结果表明，LPS 处理显著诱导多种炎症因子的表达水平，包括肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β、IL-6 和 IL-8，并增加 ATDC5 细胞中 Let-7a 的表达水平。通过 TargetScan 和双荧光素酶报告基因检测，鉴定出白细胞介素 6 受体（IL-6R）是 Let-7a 的直接靶标。随后，细胞计数试剂盒-8 和流式细胞术分析表明，LPS 处理的 ATDC5 细胞中 Let-7a 抑制剂可显著促进细胞活力并减少细胞凋亡，而用小干扰（si）RNA-IL-6R 转染可逆转这些作用。ELISA 用于检测 LPS 处理后 ATDC5 细胞中炎症因子的表达。此外，RT-qPCR 和 Western blot 用于检测 IL-6R 和 STAT3 的 mRNA 和蛋白表达水平。结果表明，Let-7a 抑制剂显著降低 ATDC5 细胞中 TNF-α、IL-1β、IL-6 和 IL-8 的表达水平，而用 siRNA-IL-6R 转染可逆转这种作用。此外，RT-qPCR 和 Western blot 检测结果表明，Let-7a 抑制剂显著增加 IL-6R 和磷酸化 STAT3 的表达水平，而用 siRNA-IL-6R 转染可逆转这种作用。综上所述，Let-7a 抑制剂增加 LPS 处理的 ATDC5 细胞的增殖，减少凋亡并抑制炎症反应。本研究为 OA 治疗提供了一个新的潜在治疗靶点。
