Department of Orthopaedic Surgery, Chang Gung Memorial Hospital, No 5, Fu-Hsing Street 333, Taoyuan, Taoyuan, Taiwan.
College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Arthritis Res Ther. 2019 Jan 31;21(1):42. doi: 10.1186/s13075-019-1830-1.
The expression of both high-mobility group box 1 (HMGB1) and receptor for advanced glycation end-products (RAGE) is upregulated in degenerated discs. HMGB1 is known to function as a coupling factor between hypoxia and inflammation in arthritis, and this inflammatory response is modulated by microRNAs (miRNAs), with miR-107 expression downregulated during hypoxia. In this study, we investigated the regulation of the miR-107/HMGB1/RAGE pathway in degenerated nucleus pulposus cells (NPCs) after hyperbaric oxygen (HBO) treatment.
NPCs were separated from human degenerated intervertebral disc tissues. The control cells were maintained in 5% CO/95% air, and the hyperoxic cells were exposed to 100% O at 2.5 atmospheres absolute. MiRNA expression profiling was performed via microarray and confirmed by real-time PCR, and miRNA target genes were identified using bioinformatics and luciferase reporter assays. The cellular protein and mRNA levels of HMGB1, RAGE, and inducible nitric oxide synthase (iNOS) were assessed, and the phosphorylation of MAPK (p38MAPK, ERK, and JNK) was evaluated. Additionally, cytosolic and nuclear fractions of the IκBα and NF-κB p65 proteins were analyzed, and secreted HMGB1 and metalloprotease (MMP) levels in the conditioned media were quantified.
Using microarray analyses, 96 miRNAs were identified as upregulated and 66 downregulated following HBO treatment. Based on these results, miR-107 was selected for further investigation. Bioinformatics analyses indicated that the 3' untranslated region of the HMGB1 mRNA contained the "seed-matched-sequence" for hsa-miR-107, which was validated via dual-luciferase reporter assays. MiR-107 was markedly induced by HBO, and simultaneous suppression of HMGB1 was observed in NPCs. Knockdown of miR-107 resulted in upregulation of HMGB1 expression in HBO-treated cells, and HBO treatment downregulated the mRNA and protein levels of HMGB1, RAGE, and iNOS and the secretion of HMGB1. In addition, HBO treatment upregulated the protein levels of cytosolic IκBα and decreased the nuclear translocation of NF-κB in NPCs. Moreover, HBO treatment downregulated the phosphorylation of p38MAPK, ERK, and JNK and significantly decreased the secretion of MMP-3, MMP-9, and MMP-13.
HBO inhibits pathways related to HMGB1/RAGE signaling via upregulation of miR-107 expression in degenerated human NPCs.
高迁移率族蛋白 B1（HMGB1）和晚期糖基化终产物受体（RAGE）的表达在退变的椎间盘组织中上调。HMGB1 已知在关节炎中作为缺氧和炎症之间的偶联因子起作用，并且这种炎症反应受到 microRNAs（miRNAs）的调节，miR-107 的表达在缺氧时下调。在这项研究中，我们研究了高压氧（HBO）治疗后退变核髓细胞（NPC）中 miR-107/HMGB1/RAGE 通路的调节。
从人退变椎间盘中分离 NPC。对照细胞在 5%CO/95%空气下维持，而高氧细胞在 2.5 个大气压下暴露于 100%O。通过微阵列进行 miRNA 表达谱分析，并通过实时 PCR 进行确认，使用生物信息学和荧光素酶报告基因测定鉴定 miRNA 靶基因。评估 HMGB1、RAGE 和诱导型一氧化氮合酶（iNOS）的细胞蛋白和 mRNA 水平，并评估 MAPK（p38MAPK、ERK 和 JNK）的磷酸化。此外，分析 IκBα 和 NF-κB p65 蛋白的胞浆和核部分，并定量测定条件培养基中 HMGB1 和金属蛋白酶（MMP）的水平。
通过微阵列分析，发现 HBO 处理后有 96 个 miRNA 上调，66 个 miRNA 下调。基于这些结果，选择 miR-107 进行进一步研究。生物信息学分析表明，HMGB1 mRNA 的 3'UTR 含有 hsa-miR-107 的“种子匹配序列”，通过双荧光素酶报告基因测定进行了验证。miR-107 被 HBO 明显诱导，并且在 NPC 中同时观察到 HMGB1 的抑制。在 HBO 处理的细胞中，miR-107 的敲低导致 HMGB1 表达上调，并且 HBO 处理下调 HMGB1、RAGE 和 iNOS 的 mRNA 和蛋白水平以及 HMGB1 的分泌。此外，HBO 处理上调 NPC 中胞浆 IκBα 的蛋白水平，并减少 NF-κB 的核转位。此外，HBO 处理下调 p38MAPK、ERK 和 JNK 的磷酸化，并显著减少 MMP-3、MMP-9 和 MMP-13 的分泌。
HBO 通过上调退变人 NPC 中 miR-107 的表达抑制与 HMGB1/RAGE 信号通路相关的途径。
