Department of Traditional Chinese Medicine, Yan'An People's Hospital, No. 57, Qilipu Street, Baota District, Yan'an City 716000, Shaanxi Province, China.
Department of Rheumatology and Immunology, The Second Affiliated Hospital of The Fourth Military Medical University, No. 569, Xinsi Road, Xi'an City 710038, Shaanxi Province, China.
Biosci Rep. 2019 Jul 8;39(7). doi: 10.1042/BSR20190700. Print 2019 Jul 31.
The present study aimed to investigate the regulatory roles of miR-142-3p on the aggressive phenotypes of rheumatoid arthritis (RA) human fibroblast-like synoviocytes (RA-HFLSs), and reveal the potential mechanisms relating with nuclear factor-κB (NF-κB) signaling. miR-142-3p expression was detected in RA synovial tissues and RA-HFLSs by quantitative real-time PCR (qRT-PCR) and Northern blot analysis. RA-HFLSs were transfected with miR-142-3p inhibitor and/or treated with 10 µg/l tumor necrosis factor α (TNF-α). The viability, colony formation, apoptosis, migration, invasion, and the levels of interleukin (IL)-6, and matrix metalloproteinase 3 (MMP-3) were detected. The mRNA expressions of B-cell lymphoma-2 (Bcl-2), Bax, Bad, IL-6, and MMP-3 were detected by qRT-PCR. Moreover, the expression of Bcl-2, IL-1 receptor-associated kinase 1 (IRAK1), Toll-like receptor 4 (TLR4), NF-κB p65, and phosphorylated NF-κB p65 (p-NF-κB p65) were detected by Western blot. The interaction between IRAK1 and miR-142-3p was identified by dual luciferase reporter gene assay. MiR-142-3p was up-regulated in RA synovial tissues and RA-HFLSs. TNF-α activated the aggressive phenotypes of RA-HFLSs, including enhanced proliferation, migration, invasion, and inflammation, and inhibited apoptosis. miR-142-3p inhibitor significantly decreased the cell viability, the number of cell clones, the migration rate, the number of invasive cells, the contents and expression of IL-6 and MMP-3, and increased the apoptosis rate and the expressions of Bax and Bad, and decreased Bcl-2 expression of TNF-α-treated RA-HFLSs. MiR-142-3p inhibitor significantly reversed TNF-α-induced up-regulation of IRAK1, TLR4, and p-NF-κB p65 in TNF-α-treated RA-HFLSs. Besides, IRAK1 was a target of miR-142-3p. The down-regulation of miR-142-3p inhibited the aggressive phenotypes of RA-HFLSs through inhibiting NF-κB signaling.
本研究旨在探讨 miR-142-3p 对类风湿关节炎（RA）成纤维样滑膜细胞（RA-HFLS）侵袭表型的调控作用，并揭示与核因子-κB（NF-κB）信号相关的潜在机制。采用实时定量 PCR（qRT-PCR）和Northern blot 分析检测 RA 滑膜组织和 RA-HFLS 中 miR-142-3p 的表达。用 miR-142-3p 抑制剂转染 RA-HFLS 并/或用 10μg/l 肿瘤坏死因子-α（TNF-α）处理。检测细胞活力、集落形成、凋亡、迁移、侵袭以及白细胞介素（IL）-6 和基质金属蛋白酶 3（MMP-3）的水平。qRT-PCR 检测 B 细胞淋巴瘤-2（Bcl-2）、Bax、Bad、IL-6 和 MMP-3 的 mRNA 表达。此外，Western blot 检测 Bcl-2、IL-1 受体相关激酶 1（IRAK1）、Toll 样受体 4（TLR4）、NF-κB p65 和磷酸化 NF-κB p65（p-NF-κB p65）的表达。通过双荧光素酶报告基因检测鉴定 IRAK1 与 miR-142-3p 的相互作用。miR-142-3p 在 RA 滑膜组织和 RA-HFLS 中上调。TNF-α激活 RA-HFLS 的侵袭表型，包括增强增殖、迁移、侵袭和炎症，抑制凋亡。miR-142-3p 抑制剂显著降低 TNF-α 处理的 RA-HFLS 的细胞活力、细胞克隆数、迁移率、侵袭细胞数、IL-6 和 MMP-3 的含量和表达，并增加细胞凋亡率和 Bax 和 Bad 的表达，降低 TNF-α 处理的 RA-HFLS 的 Bcl-2 表达。miR-142-3p 抑制剂显著逆转 TNF-α 诱导的 IRAK1、TLR4 和 p-NF-κB p65 在 TNF-α 处理的 RA-HFLS 中的上调。此外，IRAK1 是 miR-142-3p 的靶标。下调 miR-142-3p 通过抑制 NF-κB 信号抑制 RA-HFLS 的侵袭表型。
