[miR-515-5p targeting Toll-like receptor 4 regulates myeloid differentiation primary response gene 88/nuclear factor-kappa B pathway to inhibit apoptosis and inflammatory response of osteoarthritis chondrocytes]

Abstract

Objective: To explore the molecular mechanism of miR-515-5p in inhibiting chondrocyte apoptosis and alleviating inflammatory response in osteoarthritis (OA).

Methods: Human cartilage cell line C28/I2 was cultured in vitro and treated with 10 ng/mL interleukin 1β (IL-1β) for 24 hours to construct an in vitro OA model. C28/I2 cells were transfected with miR mimics, mimics negative control (NC), over expression (oe)-NC, and oe-Toll-like receptor 4 (TLR4), respectively, and then treated with 10 ng/mL IL-1β for 24 hours to establish OA model. Cell proliferation capacity was detected by cell counting kit 8 and 5-Ethynyl-2'-deoxyuridine, cell apoptosis and cell cycle were detected by flow cytometry, and B-cell lymphoma 2 protion (Bcl-2), Bcl-2-associated X protein (Bax), cleaved-Caspase-3, TLR4, myeloid differentiation primary response gene 88 (MyD88), p65 and phosphorylated p65 (p-p65) protein expression levels were detected by Western blot. Real-time fluorescence quantitative PCR was used to detect mRNA expression levels of miR-515-5p and TLR4, and ELISA was used to detect pro-inflammatory factor prostaglandin E2 (PGE2), tumor necrosis factor α (TNF -α), and IL-6 levels in cell supernatant. The potential binding sites between miR-515-5p and TLR4 were predicted by BiBiServ2 database, and the targeting relationship between miR-515-5p and TLR4 was verified by dual luciferase reporting assay.

Results: After the treatment of C28/I2 cells with IL-1β, the expressions of miR-515-5p and Bcl-2 protein and the proliferation ability of C28/I2 cells significantly reduced. The expression levels of Bax and cleaved-Caspase-3 protein, the levels of pro-inflammatory factors (PGE2, TNF-α, IL-6) in the supernatant of C28/I2 cells, and the apoptosis of C28/I2 cells significantly increased. In addition, the proportion of the cells at S phase and G ₂ phase decreased significantly, and the proportion of cells at G ₁ phase increased significantly, suggesting that the cell cycle was blocked after IL-1β treatment. After transfection with miR mimics, the expression level of miR-515-5p in the cells significantly up-regulated, partially reversing the apoptosis of OA chondrocytes induced by IL-1β, and alleviating the cycle arrest and inflammatory response of OA chondrocytes. After treating C28/I2 cells with IL-1β, the mRNA and protein levels of TLR4 significantly increased. Overexpression of miR-515-5p targeted inhibition of TLR4 expression and blocked activation of MyD88/nuclear factor κB (NF-κB) pathway. Overexpression of TLR4 could partially reverse the effect of miR mimics on IL-1β-induced apoptosis and inflammation of OA chondrocytes.

Conclusion: miR-515-5p negatively regulates the expression of TLR4, inhibits the activation of MyD88/NF-κB pathway and apoptosis of OA chondrocytes, and effectively alleviates the inflammatory response of the cells.

Keywords: Osteoarthritis; Toll-like receptor 4; cell apoptosis; cell cycle; chondrocyte; inflammatory response; miR-515-5p.

图 1

Inhibitory effect of overexpression of miR-515-5p on IL-1β-induced apoptosis of OA chondrocytes 过表达miR-515-5p对IL-1β诱导的OA软骨细胞凋亡的抑制作用

图 2

The promoting effect of overexpression of miR-515-5p on IL-1β-induced chondrocyte cycle in OA detected by flow cytometry 流式细胞术检测过表达miR-515-5p对IL-1β诱导的OA软骨细胞周期的促进作用

图 3

miR-515-5p targeting negative regulatory TLR4 detection miR-515-5p靶向负调控TLR4相关检测

图 4

Inhibitory effect of up-regulated TLR4 partially reversing overexpression of miR-515-5p on IL-1β-induced apoptosis of OA chondrocytes and amelioration of inflammation 上调TLR4部分逆转过表达miR-515-5p对IL-1β诱导的OA软骨细胞凋亡的抑制作用及炎症改善作用

图 5

Observation of miR-515-5p targeting TLR4 to block activation of MyD88/NF-κB signaling pathway miR-515-5p靶向TLR4阻断MyD88/NF-κB信号通路激活观测