Upregulation of miR-107 suppresses Wnt3a/β-catenin signaling in degenerated intervertebral disc cells following hyperbaric oxygen treatment—in vitro and in vivo study

doi:10.21203/rs.3.rs-1523200/v1

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Upregulation of miR-107 suppresses Wnt3a/β-catenin signaling in degenerated intervertebral disc cells following hyperbaric oxygen treatment—in vitro and in vivo study

https://doi.org/10.21203/rs.3.rs-1523200/v1

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Background: MicroRNA (miRNA) 107 expression is downregulated but Wnt3a protein and β-catenin are upregulated in degenerated intervertebral disc (IVD). We investigated mir-107/Wnt3a-β-catenin signaling in degenerated nucleus pulposus cells (NPCs) following hyperbaric oxygen (HBO) treatment.

Methods: MiRNA profiling was accomplished via microarray and real-time PCR. MiRNA targets were identified using bioinformatics and a luciferase reporter assay. After HBO treatment, the changes in Wnt3a, phosphorylated LRP6, β-catenin, cyclinD1, metalloproteinases (MMPs) were evaluated. In addition, the translocation of β-catenin was detected, the TOP flash and FOP flash activity and the secreted MMPs were quantified. Subsequently, we investigated the effects of HBO on Wnt3a /β-catenin expression and serum keratan sulfate (KS) levels in a rabbit degenerated IVD model.

Results: Our results showed ninety-six miRNAs were upregulated and 66 downregulated following HBO intervention. The 3′ untranslated region (UTR) of the Wnt3a mRNA contained the “seed-matched-sequence” for miR-107. MiR-107 was induced by HBO and a marked suppression of Wnt3a was observed simultaneously in degenerated NPCs. Knockdown of miR-107 upregulated Wnt3a expression in hyperoxic cells. HBO downregulated the protein expression of Wnt3a, phosphorylated LRP6, and cyclin D1. There was decreased TOP flash activity following HBO stimulation, whereas the FOP flash activity was not affected. HBO decreased the nuclear translocation of β-catenin and decreased the secretion of MMP 3 and 9. Moreover, rabbit serum KS levels and the area stained positive for Wnt3a and β-catenin tended to be lower in the HBO group.

Conclusions: HBO inhibits Wnt3a/β-catenin signaling related pathways by upregulating miR-107 expression in degenerated NPCs. HBO may play a protective role against IVD degeneration and could be used as a future therapeutic treatment

microRNA

Wnt3a/β-catenin signaling

intervertebral disc

degeneration

hyperbaric oxygen

The process of intervertebral disc degeneration (IDD) is believed to have a biochemical basis, with inhibition of nuclear proteoglycan synthesis and enhanced matrix degradation caused by chemical mediators that may include interleukin (IL)-1, nitric oxide (NO), and matrix metalloproteinases (MMPs) [1.2]. A common characteristic of IDD is the imbalanced synthesis and catabolism of extracellular matrix (ECM) proteins. An elevated level of MMPs has been correlated with progressive disc degeneration in humans [3] and in animal models of IDD [4].

Wnt signaling is activated upon binding of several members of the Wnt protein family to the Frizzled/low-density lipoprotein receptor-related protein 5 or 6 (Fz-LRP5/6) receptor complex. This causes β-catenin stabilization and translocation to the nucleus, where it binds to the lymphoid enhancer factor (LEF) and T-cell factor (TCF) transcription factors to activate Wnt target gene expression [5]. Wnt proteins are important IVD cell regulatory factors. β-Catenin has been reported to be upregulated in disc tissue samples from patients with disc degeneration [6]. Activation of Wnt signaling suppresses the proliferation of NPCs, promotes cellular senescence, and induces the expression of matrix MMPs 3, 7, and 9 suggesting that Wnt signaling triggers the process of IVD degeneration [7–9]. In addition, the expression of Wnt3a, GSK-3β, cyclinD1 and β-catenin was notably augmented in parallel with IDD progression [10].

MicroRNAs are endogenous non-coding small RNAs containing 20–25 nucleotides that can mediate gene regulatory events by pairing with the 3′-untranslated region (3′-UTR) of their target mRNAs and modulating their expression. MicroRNAs regulate diverse cellular processes including cell differentiation, proliferation, and apoptosis [11]. MiR640 aggravates IDD via nuclear factor κB (NF-κB) and Wnt signaling pathway [12]. Overexpression of miR-185 alleviates IDD through inactivation of the Wnt/β-catenin signaling pathway and downregulation of Galectin-3 [13]. MiR107 inhibited osteosarcoma cell proliferation and migration through inhibition of β-catenin signaling [14]. MiR103/107 targeted WNT family member 3a (Wnt3a) in preadipocytes [15]. MiR107 was reported to be downregulated in IDD [16]. However, the function of miR-107 on Wnt3a signaling regulation in IVD is not clear.

Human IVD is the largest avascular tissue in the body and it receives oxygen and glucose through the cartilage endplate [17]. The flow of nutrients and metabolites is reduced during aging and degeneration of the IVD [18]. The hypoxic state on the NP cells [19, 20] is in turn enhanced by the loss of CEP permeability during IVD degeneration [21, 22]. The oxygen concentration may show a further decrease in degenerated IVD than healthy IVD [23, 24]. Hyperbaric oxygen (HBO) treatment increased tissue/microvascular pO₂ [25]. HBO inhibits HMGB-1/RAGE signaling pathway by regulating miR-107 expression in human degenerative disc cells [26]. Because rat NPCs have been shown to exhibit increased levels of β-catenin under the hypoxic condition [13], we first investigated the effects of miR-107 on Wnt3a/β-catenin signaling and MMPs expression in human degenerated NPCs following HBO treatment in vitro.

Keratan sulfate (KS) is a major component of cartilage proteoglycans (PGs). Because the degradation of cartilage PGs in degenerative IVD caused a dramatic rise in levels of serum KS, measurement of serum KS levels may provide useful information about PG degradation occurring in an injured or degenerative IVD [27, 28]. Serum KS serves as a potential biomarker of loading of the IVD [28]. We evaluated whether these serum cartilage metabolites can be used as biomarkers for mechanical loading rabbit discs following HBO treatment.

In this study, we demonstrated that HBO increased miR-107 expression in human degenerated NPCs, as assessed via microarray analysis and confirmed by real-time PCR. We used bioinformatics to identify putative target sequences for miR-107 in the human Wnt3a and confirmed these by way of luciferase reporter assays. In addition, we investigated the effects of HBO on the expressions of miR-107/Wnt3a/LRP 6/β-catenin–mediated signaling in human degenerated NPCs. Finally, we examined the effects of HBO on Wnt3a/β-catenin expression and serum KS in a rabbit degenerated IVD model.

The experimental protocol was approved by the Human Subjects Institutional Review Board and the Institutional Animal Care and Use Committee of the Chang Gung Memorial Hospital.

Nucleus pulposus cell isolation and culture

Fresh disc tissues were harvested from 28 degenerated lumbar IVD patients undergoing spinal surgery. IVD degeneration grade was determined by evaluation of MRI. NPCs were separated from the nuclear tissue by sequential enzymatic digestion, first with 0.4% pronase (Sigma, MO) for 1 hour and subsequently with 0.025% collagenase P (Boehringer Mannheim, Germany) and 0.004% DNase II (Sigma) at 37°C overnight. After digestion, the cells were washed extensively with DMEM/F-12 and then seeded in 3 fresh flasks at a density of 5000 cells/cm² and incubated in a humidified atmosphere of 5% CO₂ and 95% air until the cells attained confluence.

Exposure to intermittent HBO

The cells were plated at 3 × 10⁵ cells per 100-mm culture dish in 10 ml of DMEM/F-12 containing 5% FBS. Control cells were maintained in 5% CO₂/ 95% air at 1 atmosphere throughout the experimental protocol. All hyperoxic cells were exposed to 100% O₂ at 2.5 ATA (atmospheres absolute) in a hyperbaric chamber (Cherng Huei Corporation, Taiwan) for 90 minutes per 48 hours. A series of 3 times of HBO treatment was used. At 24 hours after each treatment, conditioned media (CM) were collected and centrifuged at 1200 g for 5 minutes to remove debris, and then stored at − 70°C until analysis.

MicroRNA Profiling

Total RNA was extracted from cells using mirVana miRNA isolation kit (Ambion, Austin). MiRNA expression profiling was accomplished using TaqMan Human MicroRNA Array A Cards containing 384 mature human microRNAs (Applied Biosystems, USA) and an ABI 7900 real-time PCR System according to the manufacturer’s protocol. MiRNA expression profiling was performed on ten samples from five patients (with or without HBO treatment). Briefly, 3 µL of total RNA from each sample were reverse-transcribed using the Taq-Man miRNA Reverse Transcription Kit (Applied Biosystems) and the stem-loop Megaplex Primer Pool Sets. A total of 7.5 µL of reaction mixture was immediately incubated under the following conditions: 40 cycles at 16°C for 2 min, 42°C for 1 min, and 50°C for 1 s, and 85°C for 5 min. Then, 2.5 µL of the resultant Megaplex RT products were mixed with 2.5 µL of Megaplex PreAmp Primers and 12.5 µL of TaqMan PreAmp Master Mix. A total of 25 µL of the reaction mixture was incubated using the following program: 95°C for 10 min, 55°C for 2 min, and 72°C for 2 min followed by 12 cycles at 95°C for 15 s, 60°C for 4 min, and 99.9°C for 10 min. The pre-amplified cDNA was diluted with 0.1× TE (10mM Tris, pH 8.0, 1mM EDTA) to 100 µL and used for PCR. The relative miRNA expression levels were calculated by the 2^−ΔΔCt method as follows: ΔCt (test) = Ct (miRNA of interest, test) − Ct (internal reference, test), ΔCt (calibrator) = Ct (miRNA of interest, calibrator) − Ct (internal reference, calibrator), ΔΔCt = ΔCt (test) - ΔCt (calibrator). The inter-individual variability of the efficiency of our procedures was controlled by spiking of U6 snRNA. The hierarchical cluster analysis of differentially expressed miRNAs was performed using CLUSTER 3.0 [29], and the hierarchical clustering heat map was visualized by Tree View [30].

Real-time PCR.

TaqMan miRNA assays (Applied Biosystems) were used to detect the expression levels of the mature miR-107. For the reverse transcription (RT) reactions, 10 ng of total RNA were mixed with the RT primer. RT reactions were performed at 16°C for 30 min, 42°C for 30 min, 85°C for 5 min, and then maintained at 4°C. Following the RT reactions, 1.5 µl of cDNA was used for a polymerase chain reaction (PCR) using 2 µl of TaqMan primers. The PCR was conducted at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 60 s in an ABI 7900 real-time PCR system (Applied Biosystems). The quantitative PCR results were analyzed and expressed as the relative miRNA level using the U6 snRNA for normalization purposes. The fold change in the miRNA expression in each sample relative to the average expression in the control was calculated based on the threshold cycle (CT) value using the 2^−ΔΔCt method.

MiRNA target prediction and dual-luciferase reporter assays

Target Scan 7.2 and miRnalyze online software were used to analyze the putative target genes of miR-107. The 3′UTR of Wnt3a containing the miR-107 binding site was cloned into pmirGLO dual-luciferase miRNA reporter vectors (Promega, USA). A mutated 3′ UTR of Wnt3a was introduced into the potential miR-107 binding site. The reporter vectors containing the wild-type or mutant Wnt3a 3′ UTR were transfected into NPCs using Lipofectamine 3000 (Invitrogen, USA). Briefly, cells were seeded in 6-well tissue culture plates at 2 × 10⁵ cells/per well in 2.5 mL Opti-MEM (Invitrogen) at 12 h before transfection. On the day of transfection, the cells were exposed to reporter vectors- -Lipofectamine 3000 mixtures containing 2.5 µg of the luciferase reporter plasmid DNA mixture. At 8 h after transfection, the transfection medium was changed to (DMEM)/F-12 culture medium with 5% FBS and the cells were exposed to HBO. After 48 h, the transfected cells were washed with PBS and harvested using passive lysis buffer (Promega). Cell lysates (20 µL) were evaluated for luciferase activity using a Dual-luciferase reporter assay kit (Promega) in accordance with the manufacturer’s instructions.

Transfection of NPCs with anti-miRNAs and analysis following HBO intervention

NPCs were seeded into six-well plates at a density of 2 ×10⁵ cells/well in culture medium without antibiotics. The next day (day 1), cells were transfected with anti-miR-107 (100 nM; Ambion, USA) using RNAiMAX (Invitrogen, USA) and cultured in an incubator at 37°C with 5% CO₂. After 8 h of transfection, the culture medium was changed to DMEM/F-12 with 10% FBS, and the cells were exposed to HBO intervention. On days 3 and 5, the cells were re-transfected once and exposed to HBO. At 12 h after the third HBO treatment, cellular RNA was isolated using an RNeasy mini kit (Qiagen, USA) and reverse-transcribed into cDNA with the ImProm-II reverse transcription system (Promega, USA). For real-time PCR detection of Wnt 3a transcripts, cDNA was analyzed on an ABI PRISM 7900 sequence detection system using TaqMan PCR Master Mix (Applied Biosystems, USA). The cycle threshold (Ct) values were obtained, and the data were normalized to β-actin expression using the 2^−ΔΔCt method to calculate the relative mRNA levels of each target gene.

At 24 h after the third HBO intervention, cells were washed with PBS and extracted using M-PER mammalian protein extraction reagent (Thermo, USA). For immunoblotting experiments, the proteins were separated via SDS-PAGE and transferred onto nitrocellulose membranes using a protein transfer unit (Bio-Rad, USA). After blocking with 10% nonfat milk, the membranes were incubated overnight at 4°C with a 1000-fold dilution of mouse antibodies against Wnt 3a (Cell Signaling, USA). After washing, the membranes were further incubated for 2 h with 10,000-fold goat anti-mouse IgG (Calbiochem, USA) conjugated to horseradish peroxidase. The membranes were then washed and rinsed with ECL detection reagents (Amersham Pharmacia Biotech, UK). The band images were photographed using Hyperfilm (Amersham). The intensity of the staining for Wnt 3a and β-actin was quantified using an image analysis system (Image-Pro plus 5.0; Media Cybernetics, USA).

Protein extraction and Western blot analysis of Wnt3a, phosphor-LRP6, LRP6, and cyclin D1

Cells were plated at a density of 3 × 10⁵ cells per 100-mm culture dish in 10 ml of DMEM/F-12 containing 5% FBS. At 24 h after 3 times of HBO intervention, the cells were washed with PBS and extracted using M-PER protein extraction reagent (Thermo, USA). The protein content was quantitated using a protein assay kit (Pierce Biotechnology, IL), separated by 7.5% SDS-PAGE, and transferred onto membranes using a transfer unit (Bio-Rad, USA). After blocking, the membranes were incubated with 1000-fold diluted rabbit antibodies against Wnt 3a, phosphor-LRP6 (Cell Signaling), LRP6 (Abcam, UK), cyclin D1/2 (Merck, Germany), mouse antibodies against β-catenin (Millipore, CA) and β-actin (Millipore). After washing, the membranes were further incubated for 2 h with 10,000-fold goat anti-mouse IgG (Calbiochem, USA) or goat anti-rabbit IgG (Millipore) conjugated to horseradish peroxidase. The membranes were then washed and rinsed with ECL detection reagents (Millipore). The bands were photographed using ECL Hyperfilm (Amersham Pharmacia UK) and their intensity was quantified using an image-analysis system (Image-Pro plus 5.0).

Preparation of cytosolic and nuclear fractions for β-catenin detection

At 24 h after 3 times of HBO treatment, the cells were rinsed with PBS, treated with 0.05% trypsin, and then collected by centrifugation at 800 g. NE-PER nuclear and cytoplasmic extraction reagents (Thermo Fisher) were used to isolate cytoplasmic and nuclear extracts from the cells. The protein content was quantitated using a protein assay kit (Pierce), and separated by 7.5% SDS-PAGE to detect β-catenin (Millipore, USA) and TATA binding protein (TBP; Abcam).

TOP/FOP flash luciferase assay

The activity of the Wnt signaling pathway was detected by a Wnt signal reporter assay using the TOP/FOP TCF reporter kit (Millipore, USA). Cells were seeded in 24-well tissue culture plates at 5 × 10⁴ cells/well in 0.5 mL of Opti-MEM (Invitrogen) at 12 h before transfection. On the day of transfection (day 1), 900 ng of the TOP flash or FOP flash construct (Millipore) together with 100 ng of the pGL4.74 [hRluc/TK] plasmid (Promega) was used to transfect the cells in each well. The pGL4.74 [hRluc/TK] plasmid containing the Renilla luciferase gene was used as an internal control for normalizing the transfections. Transient transfections using Lipofectamine LTX and PLUS reagent (Invitrogen) were performed according to the manufacturer's instructions. Eight hours after transfection, the transfection medium was changed to DMEM/F-12 containing 5% FBS, and the cells were exposed to HBO. On days 3 and 5, the cells were re-transfected once and exposed to HBO as described above. After an additional 24 h of culturing, the cells were washed with PBS and harvested using 100 µL/well of passive lysis buffer (Promega). The cell lysates (20 µL) were evaluated for luciferase activity using a Dual-Luciferase Reporter Assay Kit (Promega). Luciferase activity was measured according to the manufacturer's instructions.

RNA extraction and real-time PCR detection of Wnt3a, MMP-3, and MMP-9

At 24 h after 3 times of HBO treatment, cellular RNA was isolated using an RNeasy mini kit (Qiagen, CA) and reverse-transcribed into cDNA with the ImProm-II reverse transcription system (Promega). For real-time PCR detection of Wnt3a, MMP-3, and − 9 RNA transcripts, cDNA was analyzed on an ABI PRISM 7900 sequence detection system using TaqMan PCR Master Mix (Applied Biosystems). The cycle threshold (Ct) values were obtained, and the data were normalized to GAPDH expression using the 2^−ΔΔCt method to calculate the relative mRNA levels of each target gene.

MMPs ELISA assay

The levels of MMP-3 and − 9 in condition media (CM) after hyperbaric or normobaric treatments were determined using a commercial immunoassay kit (Quantikine Human MMP-3 and MMP-9; R&D System). At 24 hours after each treatment, 200 µL of CM was sampled and analyzed according to the manufacturer’s instructions. All measurements were performed in duplicate. The results were normalized to µg DNA. The DNA content was determined using DNAzol reagent (DNAzol; Invitrogen, MD) according to the manufacturer’s instructions.

Effect of HBO on rabbit IVD degeneration model

Rabbit IVD degeneration model

We developed the external compression device that was used by Kroeber et al [31] to create intervertebral disc degeneration in rabbit model. Briefly, six New Zealand rabbits (skeletally mature, 3.2 ± 0.4 kg) were anesthetized by injection of a 5 ml mixture (1:1) of Zoletil and Rompum. With the six rabbits under general anesthesia, through a dorsal approach to the lumbar spine, the custom-made external loading device was attached using four stainless steel pins inserted into the vertebra body L4 and L5 parallel to the adjacent study disc by the use of a variable-speed electric drill. After the wound was closed, axial stress to the disc was immediately created by a calibrated spring within the loading device so as to produce a disc compressive force in animals.

Effect of HBO on degenerated rabbit IVD

Six IVD degenerated rabbits were used and divided into two groups: (I) HBO group, which included 3 rabbits exposed to HBO with 100% oxygen at 2.5 ATA for 1.5 h daily, 5 days in a week after surgery in a HBO animal chamber (Perry Baromedical Corporation, Riviera Beach, FL) and (II) Control group, housed in cages containing normal air. After 4 weeks of mechanical loading, the custom-made external loading devices were removed and Safranin-O staining was used to identify disc degeneration.

Antigenic KS detection in blood serum

At intervals of 1, 2 and 4 weeks after operation, venous blood was obtained from the rabbit ears in both the control and HBO groups. Blood serum was obtained after centrifugation and analyzed for agKS (antigenic KS, bearing the 1/20/5-D-4 epitope) content by competitive indirect ELISA [25, 54]. Briefly, after an inhibition step in which the 1/20/5-D-4 monoclonal antibody was allowed to interact with agKaS in a diluted sample of serum, the mixture was placed in the well of a microtiter plate coated with chondroitinase ABC treated, agKS-containing aggrecan. After rinsing, the mixture was incubated with a solution of peroxidase coupled anti-mouse IgG. Then, after further rinses, the new mixture was incubated with a solution containing the substrate for peroxidase. The absorbance of the colored product was read at 490 nm. agKS concentration was calculated by comparing this absorbency value with that generated using serial dilutions of an international standard of purified bovine KS (Sigma, USA). The KS standard was used at concentrations ranging from 6.25 to 200 ng/ml.

Morphological observation and immunohistochemically examination for Wnt 3a and β-catenin

The degenerated IVD was sent for immunohistochemically analysis. Tissue blocks were fixed in 10% formalin, decalcified with 20% EDTA, and embedded in paraffin. Five-micron sections were cut and stained with Safranin-O. Tissue sections were deparaffinized and incubated with proteinase K (25 µg/ml, Sigma) for 60 min at 37°C. Endogenous peroxidase activity was blocked with 1% hydrogen peroxide. The presence and distribution of Wnt 3a and β-catenin was determined using antibodies to detect Wnt 3a (1:200, Santa Cruz Biotechnology) and β-catenin (1:40 dilution, BD Transduction Laboratories) with exposure at 4°C overnight. Subsequently, a HRP linking 2° Ab was used for 30 min. Bound immunoglobulin was detected using a Dako REAL Envision Detection System (Dako, Carpinteria, CA) and 0.1% methyl green (Dako) was used for counterstaining.

Statistical Analysis

Each human disc yield one sample. The control and HBO samples separated from the same (Control group: without HBO treatment) so we used paired t-test to analyze the control/HBO ratio in this study. Data were represented as mean ± standard division (SD). The p-values for the paired Student's t-test were performed using the SPSS software package (Version 12.0, Chicago: SPSS Inc.). A p-value of < 0.05 was considered statistically significant.

Heat maps of miRNA expression in degenerated NPCs following HBO treatment

As shown in Fig. 1a, there were 116 miRNAs upregulated and 67 downregulated by at least 1.5-fold following HBO treatment (n = 5). Among these, miR-107 was chosen for further investigation (Fig. 1b) as previous studies revealed that miR-107 mediates an inflammation response [32] and its expression is lower in IDD tissue than in normal IVD [16].

HBO increased miR-107 expression in degenerated NPCs

HBO increased miR-107 expression in degenerated NPCs (HBO/control, 6.44 ± 1.67 fold, **p < 0.01, n = 5; Fig. 1c). These results suggested that miR-107 might play an important role in inhibiting the progression of IDD in degenerated NPCs following HBO treatment.

Seed sequence of miR-107 in the 3’ UTR of the Wnt3a mRNA

To investigate the potential molecular targets of miR-107, we screened for putative target genes of miR-107 using (a) Target Scan 7.2 and (b) miRnalyze online software. We found that Wnt3a, an important stimulator of canonical pathway, was likely a direct target of miR-107, as the 3′ UTR of Wnt3a contained a potential binding element for miR-107 with a 7-nt match to the miR-107 seed region (Figs. 2a and 2b). Additionally, cross-species conservation of the miR-107 seed sequence in the 3’ UTR of the Wnt3a mRNA was confirmed by the Target Scan algorithm (Fig. 2c). These results indicated that hsa-miR-107 might target the Wnt3a mRNA by directly recognizing its seed-matched sequence present in the 3’ UTR.

Wnt3a is a direct target of miR-107

To validate the direct targeting of Wnt3a by miR-107, the wild type (WT) or a mutant variant (Mut) of the Wnt3a 3′ UTR containing the target sequence was cloned into a dual-luciferase reporter vector (Fig. 3a). Overexpression of miR-107 after HBO treatment significantly inhibited luciferase activity of the WT Wnt3a 3′ UTR (HBO/control, 0.65 ± 0.09 fold, p**< 0.01), whereas mutation of the miR-107 binding sites abolished this inhibitory effect of miR-107 (1.01 ± 0.09 fold, p > 0.05) in the degenerated NPCs (Fig. 3b). These observations support the conclusion that Wnt3a is a target gene of miR-107 following HBO intervention.

We next examined the expression of Wnt3a in NPCs transfected with an anti-miR-107 construct. As shown in Fig. 3c, HBO decreased the mRNA expression of Wnt3a (HBO/control, 0.37 ± 0.05 fold, ***p < 0.001, n = 4), whereas transfection with anti-miR-107 increased the mRNA level of Wnt3a in NPCs after HBO intervention (HBO/control, 0.72 ± 0.09 fold, *p < 0.05, n = 4). Western blot analysis was performed to examine the protein level of Wnt3a (Fig. 3d), and the results indicated that HBO treatment led to a significant decrease in the protein level of Wnt3a (HBO/control, 0.48 ± 0.10 fold, **p < 0.01, n = 4), whereas knockdown of miR-107 partly reversed Wnt3a protein expression in NPCs following HBO intervention (HBO/control, 0.75 ± 0.09 fold, *p < 0.05, n = 4). These data indicated that Wnt3a was negatively mediated by miR-107 at the post-transcriptional level in NPCs after HBO treatment, as overexpression of miR-107 after HBO treatment significantly inhibited the mRNA (Fig. 3c) and protein (Fig. 3d) expression of Wnt3a in these cells.

Effects of HBO on Wnt3a, LRP6 phosphorylation, β-catenin translocation and cyclin D1 expression,

The Wnt3a /β-catenin pathway is initiated through Wnt3a interaction with Frizzled and LRP5/6. LRP5/6 are single transmembrane–spanning cell surface receptors for Wnt-ligands. We determined Wnt3a/β-catenin-signaling kinetics by detecting phosphorylated LRP6 and β-catenin upon HBO stimulation. The western blot data showed that the protein level of Wnt3a (HBO/control, 54.2% ± 11.1%, *p < 0.05, n = 3) and phosphorylated LRP6 (HBO/control, 35.6% ± 5.0%, p** < 0.01, n = 3) was downregulated after culturing for 5 d with HBO intervention (Fig. 4a). In addition, the suppression of the Wnt3a pathway resulted in a downregulated expression of Wnt3a target gene, the protein cyclin D1 (HBO/control, 59.5% ± 9.5%, *p < 0.05, n = 3; Fig. 4a). Next, we examined the influence of HBO on β-catenin nuclear translocation. After the extraction from cytoplasm and nucleus, the protein levels of total β-catenin (HBO/control, 44.0% ± 3.6%, **p < 0.01, n = 3; Fig. 4a) and that of β-catenin in the nuclear fractions (HBO/control, 30.4% ± 6.8%, **p < 0.01, n = 3; Fig. 4b) were downregulated after HBO treatment. HBO decreased the translocation of β-catenin from the cytosol into the nucleus and demoted the expression of cyclin D1.

Effects of HBO on the activity of the Wnt/β-catenin signaling

To evaluate the effects of HBO on the activity of the Wnt/β-catenin signaling, we measured the activity of both TOP flash (containing the wild-type TCF binding sites) and FOP flash (mutant TOP flash) in NPCs following HBO treatment. Figure 5 shows that there was decreased TOP flash activity following HBO stimulation (control: 1.05 ± 0.07-fold vs. HBO: 0.52 ± 0.09-fold, **p < 0.01, n = 3), whereas the FOP flash activity (control: 1.03 ± 0.02-fold vs. HBO: 1.06 ± 0.06-fold, p > 0.05, n = 3) was not affected. HBO decreased the activity of the Wnt/β-catenin signaling.

Effects of HBO on the mRNA expression of MMPs

MMPs are transcriptional targets of the canonical Wnt pathway. At 24 h after 3 times of HBO treatment, Wnt-target MMPs such as MMP-3 (HBO/control, 45.0% ± 17.3%, ***p < 0.001, n = 4) and MMP-9 (HBO/control, 27.0% ± 9.1%, ***p < 0.001, n = 4, Figure. 6a) were transcriptionally downregulated in the degenerated NPCs following HBO treatment.

Effects of HBO on MMPs secretion

Figure 6b, 6c, and Table 1 present the effects of HBO on MMP-3 and − 9 protein secretion by degenerated NPCs. HBO significantly decreased the secretion of MMP-3 (Fig. 6b, Table 1) and − 9 (Fig. 6c, Table 1) by NPC after 2 or 3 times of HBO intervention. HBO decreased the Wnt signaling, which contribute to the regulation of MMPs secretion.

Table 1

**Effect of HBO on the extracellular release of MMPs by degenerated NPCs**.
MMP-3	Control	HBO	p-value
1st treatment	283.4 ± 28.2	260.5 ± 26.4	p > 0.05
2nd treatment	299.3 ± 38.8	227.3 ± 16.6	*p < 0.05
3rd treatment	316.3 ± 38.7	173.9 ± 19.1	***p < 0.001
MMP-9	Control	HBO	P-value
1st treatment	115.5 ± 7.1	103.6 ± 10.6	p > 0.05
2nd treatment	113.9 ± 12.0	79.0 ± 8.3	**p < 0.01
3rd treatment	121.7 ± 14.7	61.3 ± 8.9	***p < 0.001
Differences between control and HBO groups were analyzed using the Student t-test. A p-value < 0.05 was considered statistically significant.

Effects of HBO on degenerated rabbit IVD

External axial loading in the rabbit IVD was used to induce disc degeneration (Figure. 7). After loading, disorganization of the architecture of the annulus occurred. Safranin-O staining showed that mechanical loading induced severe proteoglycan (PG) loss, calcification of the IVD, and loss of the demarcation between the NP and AF (Fig. 8a). HBO suppressed the PG loss induced by mechanical loading (Fig. 8b) as compared with control group (Fig. 8a).

The serum keratan sulfate (KS) levels in both groups are shown in Fig. 8c and Table 2. No significant differences were noted in pre-operative serum KS levels between the control and HBO groups (p > 0.05, n = 3). After mechanical loading, serum KS levels rose sharply on week 1, and then dropped in week 2, and week 4 in the control group. Serum KS levels were also increased in the HBO group on week 1, but were significantly lower than that of the control group in 2 (*p < 0.05, n = 3) and 4 weeks (*p < 0.05, n = 3) after loading. Serum KS level was significantly increased after mechanical loading and gradually near returned to baseline after HBO treatment (Fig. 8c, Table 2).

Table 2

Effect of HBO on the rabbit’s serum KS level
KS	Control	HBO	p-value
pre-OP	121.0 ± 17.0	115.7 ± 14.7	p > 0.05
post-OP 1 Ws	171.7 ± 13.2	146.3 ± 13.6	p > 0.05
post-OP 2 Ws	162.1 ± 8.1	115.6 ± 18.8	*p < 0.05
post-OP 4 Ws	148.1 ± 9.4	105.1 ± 16.9	*p < 0.05
Differences between control and HBO groups were analyzed using the Student t-test. A p-value < 0.05 was considered statistically significant.

Effects of HBO on Wnt3a and β-catenin expression in degenerated IVD tissue

In the repaired IVD tissue, a reduced staining intensity ratio of Wnt3a (percent of positive stained area) was observed in the cytoplasm and cartilage matrix of the HBO group (Fig. 9b, 4.5% ± 0.72%) as compared to the control group (Fig. 9a, 26.2% ± 4.2% ; ***p < 0.001, n = 3). In addition, stronger expression was observed in the β-catenin positive staining intensity ratio in the control group (Fig. 9c, 3.5% ± 0.81% ) as compared to the HBO group (Fig. 9d, 1.0% ± 0.63% ; *p < 0.05, n = 3). IHC demonstrated that HBO treatment markedly suppressed Wnt3a and β-catenin expression in repaired IVD tissue.

MiR-107 regulates gene expression involved in cell division [33], hypoxic stress response [34, 37], and angiogenesis [35] in different tissues and cells. The effect of oxygen on miR-107 expression is cell type dependent. MiR-107 expression was upregulated by hypoxia in human cancer cell lines [34]. However, miR-107 expression was increased in the retina following hyperoxia treatment has also been reported [36]. Although IVD is a hypoxic tissue, the oxygen concentration showed a further decreased in degenerated IVD compared to healthy IVD [23, 24] and MiR-107 is downregulated in degenerated IVD [16]. To identify the miRNAs involved in the molecular regulation of NPCs following HBO intervention, the miRNA expression profile of NPCs was performed. In this study, miR-107 is one of the identified miRNAs upregulated in human degenerated NPCs following HBO treatment by microarray analysis (Figs. 1a and 1b) and confirmed this expression via real-time PCR (Fig. 1c). MiR-107 is downregulated during hypoxia [34], HBO increases the oxygen levels in culture media to improve hypoxia conditions and therefore upregulate the expression levels of miR-107 in degenerated NPCs.

The expression of Wnt 3a, GSK-3β, cyclinD1 and β-catenin was notably augmented in parallel with IDD progression [10]. MiR-107 is downregulated in degenerated IVD [16] and Wnt 3a was identified as a target gene of miR-107 in the present study (Figures. 2 and 3). These observations suggest that miR-107 might regulate signaling via a mechanism targeting Wnt3a. Since Wnt3a has been shown to be expressed in human and rat IVD tissue sections [7] and rat NPCs have been shown to exhibit increased levels of β-catenin under the hypoxic condition [38], we first investigated the role of the Wnt3a ligand in human degenerated NPCs following HBO treatment. The Q-PCR and Western blot analyses revealed that Wnt3a was expressed in human degenerated NPCs and that HBO significantly decreased the mRNA (Fig. 3c) and protein (Fig. 3d) expressions of Wnt3a as compared with the control cells and miR-107 inhibitors reversed the suppressive effects of HBO. The canonical Wnt/β-catenin signaling pathway is implicated in an array of cellular pathological processes including cell proliferation, apoptosis and differentiation [39]. In particular, the involvement of Wnt/β-catenin signaling in the pathogenesis of IDD has been currently revealed. However, different researches arrive at contradictory results considering the role of Wnt signaling in mediating this degenerative disease. Smolders et al reported that the downregulated Wnt signaling was involved in the early stage of IDD development in the chondrodystrophic dog model [40]. Sun et al. found that Wnt/β-catenin was suppressed by miR-532 and suppression of Wnt/β-catenin signaling remarkably boosted miR-532-induced NPC cell apoptosis [41]. Shh signaling and canonical Wnt signaling pathways are downregulated in adult IVDs and this downregulation is reversible [42]. Inversely, Hiyama et al. indicated that Wnt signaling activation retarded NPCs proliferation and promoted cell senescence. Wnt/β-catenin signals contributing to the pathogenesis of IVD degeneration. Activation of Wnt/β-catenin signaling may lead to an increased breakdown of the matrix and might cause the degeneration of NPC [7–9]. Beyond these contradictory results, the important role of Wnt signaling pathway was shown in IDD development. In the present study we noted that Wnt/β-catenin signaling was downregulated by miR-107 following HBO treatment in NPCs (Figures. 2 and 3). This implies that HBO may exert the protective effects on NPC cell through inhibition of Wnt3a/β-catenin pathway.

Wnt/β-catenin signaling triggers the process of IVD degeneration [7–9]. A key step after Wnt stimulation is the phosphorylation of the LRP6 intracellular domain, and Wnt-induced LRP6 phosphorylation has been shown to initiate Wnt/β-catenin signaling [43, 44]. Our western blot data showed that the protein level of phosphorylated LRP6 was downregulated after culturing for 5 d with HBO treatment (Fig. 4a). HBO treatment significantly decreased the expression of Wnt3a (Figs. 3c and 3d), and thus decreased the Wnt-induced LRP6 phosphorylation in the degenerated NPCs (Fig. 4a).

Oxygen reaches the NP predominantly through diffusion, thereby imposing a hypoxic state on the NP cells [19, 20], which is in turn enhanced by the loss of CEP permeability during IVD degeneration [21, 22]. Because rat NPCs was shown to exhibit increased levels of β-catenin under the hypoxic condition [38] and β-catenin protein was up-regulated in IDD tissue [6], we used HBO to improve hypoxia conditions and found that HBO resulted in decreased β-catenin protein levels in the degenerated NPCs (Fig. 4a). In addition, the protein levels of β-catenin in the nuclear fractions were downregulated after HBO treatment. HBO decreased the translocation of β-catenin from the cytosol into the nucleus (Fig. 4b), β-catenin may interact with TCF/LEF transcription factors and regulated Wnt3a target genes [5, 39] and demoted the expression of cyclin D1 (Fig. 4a).

To further evaluate the activation of β-catenin signaling, we measured the basal activity of the TOP flash reporter in the NPCs. The TOP/FOP Flash luciferase reporter assay has been developed as a reliable, substantially amplified readout of Wnt/β-catenin pathway activation. Figure 5 shows that there was decreased TOP flash activity following HBO stimulation, whereas the FOP flash activity was not affected. The activation of the TOP flash reporter is known to be specific to the Wnt3a genes [32, 45]. HBO decreased the expression of Wnt3a, which suppressed β-catenin–TCF transcriptional activity in this study. NPCs from degenerated discs have been reported to produce catabolic and inflammatory factors, including IL-1β, NO, and MMPs [1, 2]. We previously reported that HBO decreases the expression of MMP-3 in degenerated NPCs [46]. Because MMP-3 and − 9 are transcriptional targets of the canonical Wnt pathway [9, 47], we investigated the mechanisms controlling the Wnt3a/β-catenin signaling and its transcriptional activation in degenerated NPCs following HBO treatment. Our results suggested that Wnt-target MMPs such as MMP-3 and − 9 were transcriptionally downregulated in the degenerated NPCs after HBO treatment (Fig. 6a). HBO decreased Tcf-dependent transcription and suppressed the expression of Wnt-target MMPs such as MMP-3 and − 9. In addition, the protein levels of MMP-3 and − 9 (Fig. 6b) secreted by degenerated NPCs were also downregulated following HBO treatment.

Activation of Wnt/β-catenin has been reported to inhibit NPCs proliferation and trigger cell senescence thereby promoting IDD aggravation [9]. Li et al. placed rat NPCs under a pressure of 1.0 MPa and found that the Wnt/β-catenin signaling pathway was activated in a time-dependent manner [48]. Mechanical loading induced IVD degeneration and trended to upregulate the markers of Wnt signaling and β-catenin expression in IVD. In addition, mechanical loading also increased catabolic markers MMP-3 and − 13 expression in IVD [48]. In the present study, we developed the rabbit external compression model that used by Kroeber et al [31] to investigate the effects of HBO to stimulate disc regeneration (Fig. 7). Mechanical compression of the rabbit IVD induced severe PG degradation, calcification of the IVD, and loss of the demarcation between the NP and AF (Fig. 8a). Excessive loading supports upregulation of matrix-degrading enzymes and rather than a stimulus for matrix degradation [49, 50]. Mechanical compression increased catabolic markers of Wnt signaling in IVD [10]. HBO suppressed the Wnt3a/β-catenin signaling induced by mechanical loading (Figs. 9b and 9d) thus suppressed PG loss in IVD (Fig. 8b). Serum KS serves as a potential biomarker of loading of the IVD [28]. Serum KS level was significantly increased after mechanical loading and gradually near returned to baseline after HBO intervention (Fig. 8c).

Our data suggest that HBO inhibiting the Wnt3a/β-catenin signaling pathway by upregulating mir-107 expression in human degenerated disc may be feasible as a therapeutic for degenerative disc disease. However, there are some limitations in this study. Because other miRNAs may also be relevant for the regulation of Wnt signaling in NPCs following HBO treatment, transfection with anti-miR-107 only partly suppressed the effects of HBO treatment (Fig. 3). In the future, further systematic and in-depth studies investigating the Wnt signaling following HBO treatment will be conducted. The positive effects of HBO on various conditions have been studied both in animal experiments and in clinical use [51, 52]. Recently, HBO therapy in clinical patients with full endoscopic lumbar diskectomy (FELD) has been reported [53]. HBO may provide a novel therapeutic strategy for IDD. The clinical applications of HBO in degenerative IVD diseases are needed to be further investigated.

HBO inhibits Wnt3a/β-catenin signaling related pathways by upregulating miR-107 expression in degenerated NPCs. HBO may play a protective role against IVD degeneration and could be used as a future therapeutic treatment

miRNAs : microRNAs
IVD : intervertebral disc
NPCs : nucleus pulposus cells
HBO : hyperbaric oxygen
IL-1 : interleukin-1
NO : nitric oxide
MMP : metalloprotease
Wnt3a: WNT family member 3a
KS : keratan sulfate
LRP 5/6 : low-density lipoprotein receptor-related protein 5 or 6
LEF : lymphoid enhancer factor
TCF : T-cell factor
3′ UTR : 3′ untranslated region
IDD : intervertebral disc degeneration
NF‐κB : nuclear factor κB
CM : conditioned media
RT : reverse transcription
PCR : polymerase chain reaction
WT : wild-type
Mut : mutant variant

Ethics approval

The experimental protocol was approved by the Human Subjects Institutional Review Board and the Institutional Animal Care and Use Committee of the Chang Gung Memorial Hospital.

Consent to participate

Not applicable.

Availability of data and materials

All data supporting our findings are contained within the manuscript.

Competing interests

The authors declare no competing financial and non-financial interests.

Funding

This research was supported in part by grants from the National Science Council and Chang Gung Memorial Hospital, Taiwan, Republic of China (CRRPG3K0061~3).

Authors' contributions

Conception and design: LSS,NCC,USWN, and LSJ.

Acquisition, analysis and interpretation of data: YLJ, TTT,YCY,LJW, and LSC

Drafting the article:CKY,HHY,HYJ, CCA, and CYS.

Final approval of the article: LSS, NCC, USWN, LSJ, CYS, YLJ, TTT, YCY, LJW, LSC, CKY,

HHY, HYJ, and CCA.

Acknowledgements

This research was supported in part by grants from the National Science Council and Chang Gung Memorial Hospital, Taiwan, Republic of China (CRRPG3K0061~3).

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No competing interests reported.

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Upregulation of miR-107 suppresses Wnt3a/β-catenin signaling in degenerated intervertebral disc cells following hyperbaric oxygen treatment—in vitro and in vivo study

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Abstract

Figures

Background

Methods

Nucleus pulposus cell isolation and culture

Exposure to intermittent HBO

MicroRNA Profiling

Real-time PCR.

MiRNA target prediction and dual-luciferase reporter assays

Transfection of NPCs with anti-miRNAs and analysis following HBO intervention

Protein extraction and Western blot analysis of Wnt3a, phosphor-LRP6, LRP6, and cyclin D1

Preparation of cytosolic and nuclear fractions for β-catenin detection

TOP/FOP flash luciferase assay

RNA extraction and real-time PCR detection of Wnt3a, MMP-3, and MMP-9

MMPs ELISA assay

Effect of HBO on rabbit IVD degeneration model

Rabbit IVD degeneration model

Effect of HBO on degenerated rabbit IVD

Antigenic KS detection in blood serum

Morphological observation and immunohistochemically examination for Wnt 3a and β-catenin

Statistical Analysis

Results

Heat maps of miRNA expression in degenerated NPCs following HBO treatment

HBO increased miR-107 expression in degenerated NPCs

Seed sequence of miR-107 in the 3’ UTR of the Wnt3a mRNA

Wnt3a is a direct target of miR-107

Effects of HBO on Wnt3a, LRP6 phosphorylation, β-catenin translocation and cyclin D1 expression,

Effects of HBO on the activity of the Wnt/β-catenin signaling

Effects of HBO on the mRNA expression of MMPs

Effects of HBO on MMPs secretion

Effects of HBO on degenerated rabbit IVD

Effects of HBO on Wnt3a and β-catenin expression in degenerated IVD tissue

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

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