阻断 HOTAIR 通过调控 miR-222-3p/ADAM10 轴保护人软骨细胞免受 IL-1β诱导的细胞凋亡、细胞外基质降解、炎症反应和氧化应激。

Blocking HOTAIR protects human chondrocytes against IL-1β-induced cell apoptosis, ECM degradation, inflammatory response and oxidative stress via regulating miR-222-3p/ADAM10 axis.

机构信息

Department of Joint Disease, Zhengzhou Orthopaedic Hospital, Zhengzhou, China.

出版信息

Int Immunopharmacol. 2021 Sep;98:107903. doi: 10.1016/j.intimp.2021.107903. Epub 2021 Jun 27.

DOI:10.1016/j.intimp.2021.107903

PMID:34192661

Abstract

BACKGROUND

Long non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) contributes to cartilage damages including osteoarthritis (OA). While, its role and mechanism in chondrocytes is incompletely clear.

METHODS

HOTAIR, microRNA (miR)-222-3p and ADAM metalloproteinase-like domain 10 (ADAM10) expressions were detected by real-time quantitative PCR and western blotting. The interaction between miR-222-3p and HOTAIR or ADAM10 was confirmed by dual-luciferase reporter assay. Cell injury was measured by MTS method, flow cytometry, western blotting, enzyme-linked immunosorbent assay for collagen Type II, type X, sex determining region Y-box 9 (SOX9), matrix metalloproteinase (MMP)-13, interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α, and special assay kits for malondialdehyde (MDA), reactive oxygen species (ROS) and superoxide dismutase (SOD).

RESULTS

HOTAIR was highly expressed in human OA cartilages and IL-1β-induced OA model in immortalized chondrocytes (C-28/I2). Under IL-1β stress, blocking HOTAIR was responsible to high mitochondrial activity and low early apoptosis rate, accompanied with increased B cell lymphoma (Bcl)-2 and LC3B-II/I proteins, boosted IL-10 and SOD productions, suppressed cleaved caspase-3 and p62 proteins, and decreased MDA and ROS levels, as well as elevated secretions of Type II collagen, Type X collagen, SOX9, MMP-13, IL-6, and TNF-α. Moreover, miR-222-3p was a target of HOTAIR, and its overexpression and knockdown could suppress and aggravate IL-1β-induced chondrocytes injury. Furthermore, restoring ADAM10, a target gene of miR-222-3p, counteracted the protective role of miR-222-3p upregulation.

CONCLUSION

HOTAIR might contribute to IL-1β-induced chondrocytes death, inflammation, extracellular matrix degradation, and oxidative stress in OA via miR-222-3p/ADAM10 axis.

摘要

背景

长链非编码 RNA（lncRNA）HOX 转录反义 RNA（HOTAIR）参与包括骨关节炎（OA）在内的软骨损伤。然而，其在软骨细胞中的作用和机制尚不完全清楚。

方法

采用实时定量 PCR 和 Western blot 检测 HOTAIR、微小 RNA（miR）-222-3p 和 ADAM 金属蛋白酶样结构域 10（ADAM10）的表达。通过双荧光素酶报告基因实验证实 miR-222-3p 与 HOTAIR 或 ADAM10 的相互作用。采用 MTS 法、流式细胞术、Western blot、酶联免疫吸附试验（ELISA）检测 II 型胶原、X 型胶原、性别决定区 Y 盒 9（SOX9）、基质金属蛋白酶（MMP）-13、白细胞介素（IL）-6、IL-10 和肿瘤坏死因子（TNF）-α的表达，以及丙二醛（MDA）、活性氧（ROS）和超氧化物歧化酶（SOD）的特殊测定试剂盒。

结果

HOTAIR 在人 OA 软骨和永生化软骨细胞（C-28/I2）中 IL-1β诱导的 OA 模型中高表达。在 IL-1β应激下，阻断 HOTAIR 可导致线粒体活性升高和早期凋亡率降低，同时 B 细胞淋巴瘤（Bcl）-2 和 LC3B-II/I 蛋白增加，IL-10 和 SOD 产生增加，caspase-3 和 p62 蛋白减少，MDA 和 ROS 水平降低，II 型胶原、X 型胶原、SOX9、MMP-13、IL-6 和 TNF-α分泌增加。此外，miR-222-3p 是 HOTAIR 的靶标，过表达和敲低 miR-222-3p 可抑制和加重 IL-1β诱导的软骨细胞损伤。此外，恢复 miR-222-3p 的靶基因 ADAM10，可拮抗 miR-222-3p 上调的保护作用。