Clinical Laboratory Diagnostics, Tianjin Medical University General Hospital Airport Site, Tianjin 300308, P.R. China.
School of Laboratory Medicine, Tianjin Medical University, Tianjin 300070, P.R. China.
Mol Med Rep. 2019 Sep;20(3):2703-2711. doi: 10.3892/mmr.2019.10499. Epub 2019 Jul 15.
The present study explored whether miR‑145‑5p can aggravate the development and progression of rheumatoid arthritis (RA) by regulating the expression of matrix metalloproteinases (MMPs). ELISAs, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR), and western blotting were used to examine the expression levels of MMP‑1, MMP‑3, MMP‑9, and MMP‑13 in fibroblast‑like synoviocytes (FLS) from patients with RA. Levels of MMP‑1, MMP‑3, MMP‑9, and MMP‑13 were assessed in the right hind ankles of a murine collagen‑induced arthritis (CIA) model by RT‑qPCR and immunohistochemical (IHC) analysis. The effects of activation or inhibition of the nuclear factor‑κB (NF‑κB) pathway on MMPs were evaluated by RT‑qPCR and western blotting. Subcellular localization of NF‑κB p65 was visualized by confocal microscopy. Overexpression of miR‑145‑5p increased the expression of MMP‑3, MMP‑9, and MMP‑13 in RA‑FLS. Moreover, injection of a miR‑145‑5p agomir into mice increased MMP‑3, MMP‑9, and MMP‑13, as demonstrated by RT‑qPCR and IHC analysis. A chemical inhibitor that selectively targets NF‑κB (BAY11‑7082) significantly attenuated MMP‑9 expression, while it did not influence the levels of MMP‑3 and MMP‑13. Immunofluorescence analysis revealed that nuclear localization of p65 was significantly enhanced, indicating that miR‑145‑5p enhances activation of the NF‑κB pathway by promoting p65 nuclear translocation. miR‑145‑5p overexpression also significantly increased phosphorylated p65 levels; however, the levels of IkB‑a were reduced in response to this miRNA. Moreover, our results indicated that miR‑145‑5p aggravated RA progression by activating the NF‑κB pathway, which enhanced secretion of MMP‑9. In conclusion, modulation of miR‑145‑5p expression is potentially useful for the treatment of RA inflammation, by regulating the expression of MMPs, and MMP‑9 in particular, through inhibition of the NF‑κB pathway.
本研究探讨了 miR-145-5p 是否可以通过调节基质金属蛋白酶 (MMPs) 的表达来加重类风湿关节炎 (RA) 的发展和进展。酶联免疫吸附试验 (ELISA)、逆转录-定量聚合酶链反应 (RT-qPCR) 和 Western blot 用于检测 RA 成纤维样滑膜细胞 (FLS) 中 MMP-1、MMP-3、MMP-9 和 MMP-13 的表达水平。通过 RT-qPCR 和免疫组织化学 (IHC) 分析评估鼠胶原诱导关节炎 (CIA) 模型右后踝关节中 MMP-1、MMP-3、MMP-9 和 MMP-13 的水平。通过 RT-qPCR 和 Western blot 评估核因子-κB (NF-κB) 通路的激活或抑制对 MMPs 的影响。通过共聚焦显微镜观察 NF-κB p65 的亚细胞定位。miR-145-5p 的过表达增加了 RA-FLS 中 MMP-3、MMP-9 和 MMP-13 的表达。此外，向小鼠注射 miR-145-5p 激动剂通过 RT-qPCR 和 IHC 分析证明增加了 MMP-3、MMP-9 和 MMP-13。一种选择性靶向 NF-κB 的化学抑制剂 (BAY11-7082) 显著减弱了 MMP-9 的表达，而对 MMP-3 和 MMP-13 的水平没有影响。免疫荧光分析显示 p65 的核定位明显增强，表明 miR-145-5p 通过促进 p65 核易位来增强 NF-κB 通路的激活。miR-145-5p 的过表达还显著增加了磷酸化 p65 的水平；然而，这种 miRNA 导致 IkB-a 的水平降低。此外，我们的结果表明，miR-145-5p 通过激活 NF-κB 通路加重 RA 进展，从而增强 MMP-9 的分泌。总之，通过调节 MMP-9 的表达，特别是通过抑制 NF-κB 通路，调节 miR-145-5p 的表达可能有助于治疗 RA 炎症。
