Abstract
BackgroundThe 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.MethodsNPCs were separated from human degenerated intervertebral disc tissues. The control cells were maintained in 5% CO2/95% air, and the hyperoxic cells were exposed to 100% O2 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.ResultsUsing 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.ConclusionsHBO inhibits pathways related to HMGB1/RAGE signaling via upregulation of miR-107 expression in degenerated human NPCs.
Highlights
The process of intervertebral disc degeneration (IDD) is believed to have a biochemical basis that involves inhibition of nuclear proteoglycan synthesis and enhanced matrix degradation caused by chemical mediators, such as interleukin (IL)-1, nitric oxide (NO), and matrix metalloproteinases (MMPs) [1, 2]
hyperbaric oxygen (HBO) treatment increased miR-107 expression in nucleus pulposus cells (NPCs) HBO treatment increased miR-107 expression in NPCs (5.84 ± 2.04 fold, *p < 0.05, n = 4; Fig. 1c). These results indicated that miR-107 might play an important role in inhibiting the progression of IDD in NPCs after HBO treatment
We found that high-mobility group box 1 (HMGB1), an important regulator of inflammation, was likely a direct target of miR-107, as the 3′ 3′ untranslated region (UTR) of HMGB1 contained a potential binding element for miR-107 with a 7-nt match to the miR-107 seed region (Fig. 2a,b)
Summary
The process of intervertebral disc degeneration (IDD) is believed to have a biochemical basis that involves inhibition of nuclear proteoglycan synthesis and enhanced matrix degradation caused by chemical mediators, such as interleukin (IL)-1, nitric oxide (NO), and matrix metalloproteinases (MMPs) [1, 2]. MiR-10b promotes NP cell proliferation via the RhoC-Akt pathway by targeting HOXD10 during IDD [11], and miR-146a reduces IL-1–dependent inflammatory responses in intervertebral discs (IVDs) [12]. MiR-155 promotes Fas-mediated apoptosis by targeting Fas-associated protein with death domain (FADD) and caspase-3 during human IDD [13]. These findings demonstrate the direct involvement of miRNAs in the pathogenesis of degenerative disorders; the exact role of miR-107 in IVD has not been well defined. The expression of both high-mobility group box 1 (HMGB1) and receptor for advanced glycation endproducts (RAGE) is upregulated in degenerated discs. We investigated the regulation of the miR-107/HMGB1/ RAGE pathway in degenerated nucleus pulposus cells (NPCs) after hyperbaric oxygen (HBO) treatment
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
