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微小RNA-6779-5p对白细胞介素-1β诱导软骨细胞损伤的影响及机制研究

Effect and mechanism of mircoRNA-6779-5p on chondrocyte injury induced by interleukin-1β

    摘要
  • 摘要:
    目的 分析微小RNA-6779-5p(miR-6779-5p)在白细胞介素-1β(IL-1β)刺激的软骨细胞中对细胞增殖和凋亡的影响以及相关的分子机制。
    方法 采用实时荧光定量聚合酶链反应(qRT-PCR)法分析RNA的表达量。将miR-6779-5p模拟物、白细胞介素1受体相关激酶3(IRAK3)过表达载体以及各自对照转染CHON-001细胞，采用10 ng/mL的IL-1β刺激细胞。应用功能试验分析miR-6779-5p和IRAK3对IL-1β诱导的细胞损伤的影响; 采用免疫印迹试验分析核因子-κB(NF-κB)通路的相关蛋白表达; 鉴定miR-6779-5p和IRAK3的关系。
    结果 miR-6779-5p在骨关节炎患者中表达下调，而IRAK3表达上调，差异有统计学意义(P < 0.05); miR-6779-5p和IRAK3的RNA水平在不同浓度IL-1β诱导的CHON-001细胞中也有相同的表达趋势，差异有统计学意义(P < 0.05)。IL-1β处理后, CHON-001细胞的活力(100.00%对比51.00%)和EdU阳性率降低(43.00%对比25.00%), 细胞凋亡率增加(6.43%对比18.60%), 差异有统计学意义(P < 0.05), 但这些影响在miR-6779-5p过表达后缓解(细胞活力: 52.00% 对比85.00%; EdU阳性率: 25.67%对比38.67%; 细胞凋亡率: 18.70%对比12.10%), 差异均有统计学意义(P < 0.05)。miR-6779-5p靶向结合IRAK3。IRAK3表达上调在IL-1β诱导的CHON-001细胞中逆转miR-6779-5p过表达对细胞的影响，差异有统计学意义(P < 0.05)。miR-6779-5p模拟物在IL-1β诱导的软骨细胞中减少p-P65和P65比值(2.06对比1.34)以及p-IκBα和IκBα的比值(2.42对比1.42), 差异有统计学意义(P < 0.05), 但IRAK3过表达缓解了这些影响(p-P65和P65比值: 1.30对比1.88; p-IκBα和IκBα的比值: 1.45对比2.16), 差异有统计学意义(P < 0.05)。
    结论 miR-6779-5p通过抑制IRAK3/NF-κB通路的激活来挽救IL-1β诱导的软骨细胞损伤，表明miR-6779-5p可能是治疗骨关节炎的潜在靶点。

     

    Abstract:
    Objective To analyze the effects of microRNA-6779-5p (miR-6779-5p) on cell proliferation and apoptosis in chondrocyte stimulated by interleukin-1β (IL-1β) and the related molecular mechanism.
    Methods Expression level of RNA was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). CHON-001 cells were transfected with miR-6779-5p mimics, overexpression vector of interleukin-1 receptor associated kinase 3 (IRAK3) and their respective controls, and 10 ng/mL IL-1β was used to stimulate the cells. The effects of miR-6779-5p and IRAK3 on cell injury induced by IL-1β were analyzed by functional tests; the nuclear factor kappa B (NF-κB) pathway related protein expression was analyzed by Western blotting; the relationship between miR-6779-5p and IRAK3 was identified.
    Results MiR-6779-5p expression was down-regulated in osteoarthritis patients, while IRAK3 expression was up-regulated (P < 0.05). MiR-6779-5p and IRAK3 expressions also showed the same trend in CHON-001 cells induced by IL-1β at differed concentrations (P < 0.05). IL-1β treatment decreased the activity (100.00% versus 51.00%) and EdU positive rate (43.00% versus 25.00%) of CHON-001 cells but increased cell apoptosis rate (6.43% versus 18.60%) (P < 0.05), however, these effects were alleviated after miR-6779-5p overexpression (cell viability: 52.00% versus 85.00%; the positive rate of EdU: 25.67% versus 38.67%; cell apoptosis rate: 18.70% versus 12.10%, P < 0.05). In addition, miR-6779-5p targeted IRAK3. The up-regulation of IRAK3 expression could reverse the effects of miR-6779-5p overexpression on CHON-001 cells (P < 0.05). Moreover, miR-6779-5p mimics reduced the p-P65-to-P65 ratio (2.06 versus 1.34) and the p-IκBα-to-IκBα ratio (2.42 versus 1.42) in IL-1β-induced chondrocytes (P < 0.05), but IRAK3 overexpression mitigated these effects (p-P65-to-P65 ratio: 1.30 versus 1.88; p-IκBα-to-IκBα ratio: 1.45 versus 2.16, P < 0.05).
    Conclusion MiR-6779-5p can ameliorate chondrocyte injury induced by IL-1β by inhibiting the activation of IRAK3/NF-κB pathway, which suggests that miR-6779-5p might be a potential target for the treatment of osteoarthritis.

     

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