Overexpression of miR-182 inhibits ossification of ligamentum flavum cells by targeting NAMPT

Author’s Accepted Manuscript Overexpression of miR-182 inhibits ossification of ligamentum flavum cells by targeting NAMPT Qiang Zhang, Yifei Shen, Yuqing Jiang, Shujie Zhao, Dong Zhou, Nanwei Xu www.elsevier.com/locate/yexcr

PII: DOI: Reference:

S0014-4827(18)30127-7 https://doi.org/10.1016/j.yexcr.2018.03.008 YEXCR10958

To appear in: Experimental Cell Research Received date: 30 October 2017 Revised date: 5 March 2018 Accepted date: 6 March 2018 Cite this article as: Qiang Zhang, Yifei Shen, Yuqing Jiang, Shujie Zhao, Dong Zhou and Nanwei Xu, Overexpression of miR-182 inhibits ossification of ligamentum flavum cells by targeting NAMPT, Experimental Cell Research, https://doi.org/10.1016/j.yexcr.2018.03.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Overexpression of miR-182 inhibits ossification of ligamentum flavum cells by targeting NAMPT Qiang Zhang1,2,1, Yifei Shen1,1, Yuqing Jiang1, Shujie Zhao1, Dong Zhou1,*, Nanwei Xu1,* 1

Department of Orthopaedics, The Affiliated Changzhou No. 2 People’s Hospital with

Nanjing Medical University, Changzhou, Jiangsu, China 2

Department of Spine Surgery, Shanghai East Hospital, Tongji University School of

Medicine, Shanghai, China [email protected] [email protected] *

Corresponding Author: Department of Orthopaedics, Changzhou No.2 People’s Hospital, 29# Xinglongxiang, Changzhou, 213000, Jiangsu, China. * Corresponding Author. Department of Orthopaedics, Changzhou No.2 People’s Hospital, 29# Xinglongxiang, Changzhou, 213000, Jiangsu, China. Tel./fax:+86 0519 88132527.

ABSTRACT Ossification of the ligamentum flavum (OLF) is a debilitating disease resulting from the development of ectopic bone formation, which leads to the compression of the spinal cord. Nicotinamide phosphoribosyltransferase (NAMPT) was found to be upregulated and microRNA-182 (miR-182) was downregulated in OLF tissue. We investigated the effects of NAMPT and miR-182 expression in OLF cells and the influence on proteins associated with osteogenic differentiation. MiR-182 overexpression inhibited NAMPT, RUNX2, OCN and OPN mRNA and protein expression in OLF cells. Alkaline phosphatase (ALP) assay and Alizarin red staining confirmed reduced levels of osteogenic differentiation and mineralized nodule formation. Knockdown of NAMPT and the NAMPT inhibitor FK866 also inhibits RUNX2, OCN and OPN mRNA expression and protein levels, whereas overexpression of NAMPT promotes the expression of RUNX2, OCN and OPN and 1

These authors contributed equally to this paper.

the generation of bone nodules. Dual-luciferase reporter assays revealed that miR-182 directly targets NAMPT and downregulates its expression. Transfection of OLF cells with miR-182 downregulated NAMPT and suppressed the regulation of RUNX2, OCN, and OPN by NAMPT overexpression. Overall, these data demonstrate that miR-182 suppresses OLF by downregulating NAMPT.

Keywords: Ossification, Ligamentum flavum, NAMPT, miR-182, RUNX2, OCN, OPN

1. Introduction The ligamenta flava are spinal ligaments that connect laminae of adjacent vertebrae to form the posterior wall of the spinal canal [1, 2]. The usual composition of the ligaments are elastic and connective tissue fibers but ossification results in the development of ectopic bone formation leading to the compression of the spinal cord and its roots [3, 4]. Ossification of the ligamentum flavum (OLF) can occur as a consequence of mechanical stress, diffuse idiopathic skeletal hyperostosis, and metabolic diseases such as hypoparathyroidism, and X-linked hypophosphatemia [5-7]. When the spine is subjected to mechanical stress, ossification is accelerated due to the induction of collagen synthesis mediated by transforming growth factor-1 (TGF-1) [8]. Removal of ossified tissue by posterior decompressive laminectomy is often unsuccessful owing to recurrent ossification and surgical complications [9-11]. Surgery is particularly difficult in patients with dural ossification whereby OLF and dura mater fuse to create one inseparable bony tissue [12]. Therefore, less invasive methods involving molecular techniques could be an effective alternative. Genes related to osteogenic differentiation are believed to be associated with OLF [13, 14]. OLF tissue is characterized by the expression of osteocalcin (OCN) and collagen type II, which are markers of osteoblasts and chondrocytes, respectively [15]. Other genes involved in osteogenic differentiation include bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (RUNX2) and osteopontin (OPN) [13, 16, 17]. Upregulated mRNA expression of types I, V, XI collagen and OCN has

been observed in ligamentum flavum cultures treated with interleukin (IL)-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO) indicating that inflammatory cytokines may contribute to ossification [18]. Degenerated, herniated intervertebral disks are thought to cause hypertrophy and OLF through an inflammatory response [19]. Supernatant from herniated intervertebral disks with elevated levels of IL-1α, IL-6, TNF-α, PGE2, and NO, was able to increase DNA synthesis and upregulate mRNA expression of types III, XI collagen and OCN in ligamentum flavum cells [19]. Several microRNAs (miRNAs) have been reported to regulate osteogenic differentiation and hypertrophy in ligamentum flavum cells [20-23]. Fibrosis and hypertrophy are promoted in ligamentum flavum tissue by miR-21 through the activation of IL-6 expression [20]. MiR-132-3p inhibits osteogenic differentiation of ligamentum flavum by downregulating forkhead box O1 (FOXO1), growth differentiation factor 5 (GDF5) and SRY-box 6 (SOX6)[21]. Collagen I and III levels are increased in fibroblasts with higher expression of miR-155, indicating it could contribute to the pathogenesis of ligamentum flavum hypertrophy [22]. In contrast, miR-221 reduces mRNA and protein expression of collagens I and III in fibroblasts [23]. In a previous study using microarray analysis, we found that miR-182 was downregulated in intervertebral disc degeneration [24]. Kim et al. reported that miR-182 inhibits osteoblast proliferation and differentiation by downregulating FoxO1[25]. Moreover, Wang reported that long noncoding RNA POIR is a competing endogenous RNA (ceRNA) for miR-182, leading to the derepression of its target gene FoxO1, which increased osteogenic differentiation in periodontitis patients [26]. Our analysis of samples from patients with OLF showed that nicotinamide phosphoribosyltransferase (NAMPT) was upregulated and miR-182, a miRNA involved in cell differentiation [27], was downregulated in OLF compared with non-OLF tissues. NAMPT is a rate-limiting enzyme in the NAD biosynthetic pathway that converts nicotinamide to nicotinamide mononucleotide and seems to have multiple functions in mammals [28-31]. NAMPT is believed to have an intracellular and extracellular form [30]. Intracellular NAMPT is an essential enzyme in the NAD

biosynthetic pathway whereas extracellular NAMPT is thought to act as a cytokine [32]. Li et al. reported that an increased expression of NAMPT was associated with higher NAD concentration and Sirt1 activity. NAMPT knockdown or addition of its specific inhibitor FK866 leads to lower intracellular NAD concentration and a decline in

osteogenesis

[33].

NAMPT

was

progressively

elevated

during

bone

marrow-derived mesenchymal stem cells osteogenic differentiation and may serve as the marker for osteoblast differentiation [34].

Ling reported that NAMPT deficient

mice decreased the osteoblast differentiation in bone marrow stromal cells and knockdown NAMPT attenuated the increase of RUNX2 promoter acetylation associated with osteoblast differentiation [35]. Because of its association with osteoblast differentiation and the fact it was upregulated in OLF tissue, we selected NAMPT for further study. We have also discovered that miR-182 targets the NAMPT 3′-UTR using TargetScan. In this study, we assess the miRNA-182 and 3′-UTR binding site of NAMPT by a Dual-Luciferase reporter assay performed in OLF cells. In addition, we analyzed the impact of NAMPT and miR-182 on the expression of genes involved in osteogenic differentiation and measured the formation of ossification in ligamentum flavum cells.

2. Materials and methods 2.1 Specimens Ligamentum flavum tissues were harvested aseptically from twelve OLF patients during surgery to decompress the spinal cord for myeloradiculopathy. Ligamentum flavum tissue from ten non-OLF patients who received surgery for thoracic spine trauma was used as a control. The presence of ossification was preoperatively diagnosed by X-ray and confirmed by histological examination. This study was approved by the Ethics Committee of the Changzhou No.2 People’s Hospital and informed consent was obtained from each patient.

2.2 Ligamentum flavum cell culture Cells were isolated from the ligamentum flavum tissues as described previously

[36]. After washing the specimens in phosphate-buffered saline (PBS), surrounding bone, fat, and connective tissue were removed. The ligaments were minced with microdissection scissors into pieces of approximately 0.5 mm3 under aseptic conditions and washed twice with PBS. Collagenase-treated specimens were incubated at 37°C in a humidified atmosphere, air 95%, CO2 5% for 2–3 weeks in DMEM/F-12 containing 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, until cells migrated from ligaments to become a monolayer. Medium was changed at two-day intervals. OLF and non-OLF cells at the third passage were used for experiments.

2.3 Reverse transcriptase-PCR Total RNA was extracted for cultured non-OLF and OLF cells and tissues using Trizol reagent (Invitrogen, Carlsbad, USA) following manufacturer’s instructions and RNA molecules smaller than 200 nucleotides in size were purified using the mirVana miRNA isolation kit (Ambion, Austin, USA) according to the manufacturer’s instructions, as described previously [37]. Complementary DNA (cDNA) was synthesized using the M-MLV Kit (Life Technologies, USA). QRT-PCR was carried out using the Platinum SYBR Green qPCR SuperMix-UDG kit (Life Technologies) on an ABI Prism 7500 PCR system (Applied Biosystems, USA). Data were normalized to GAPDH. QRT-PCR analysis of mature miR-182 was performed using the TaqMan MicroRNA Assay Kit (Applied Biosystems), the expression level of mature miR-182 was determined by real-time PCR analysis following stem-loop RT and data were normalized to U6 snRNA. All primers are listed in Table 1. Each test was carried out in triplicate. Relative expression was calculated using the 2−ΔΔCt method [38].

2.4 Western blotting After washing in cold PBS, ligamentum flavum cells were lysed with RIPA lysis buffer (Beyotime Bio, Haimen, China) and lysates were applied to 10% SDS-PAGE or for osteocalcin analysis on Tricine-SDS-PAGE. Proteins separated in acrylamide

gels were transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were probed with primary antibodies overnight at 4 °C, the primary antibodies were NAMPT antibody (ab58640, 1:500 dilution) (Abcam, Austin, TX, USA), RUNX2 (ab23981 1:500 dilution), OCN (ab93876, 1:1000 dilution), OPN (ab8448, 1:500 dilution) at room temperature for 2 h. Immunolabeling was detected using Pierce ECL Western Blotting Substrate (Pierce Biotechnology). Protein expression was visualized and quantified by Image J software.

2.5 Alizarin red staining To determine mineralization of extracellular matrix, Alizarin red staining was carried out as described previously [39]. Cells were first washed with PBS and then fixed with ice-cold 70% ethanol for 60 min. Washed cells were then stained with 40 mM Alizarin red (Sigma-Aldrich, St. Louis, MO, USA), pH 4.2, for 30 min at room temperature. Cells were rinsed five times with distilled water and then photographed under a microscope (IX71, Olympus, Tokyo, Japan).

2.6 Alkaline phosphatase activity assay Bone formation was measured using an ALP assay [39]. Cell extract was prepared with RIPA lysis buffer. The ALP activity in cell lysates was assayed at the end of the incubation with ALP substrate buffer containing the soluble substrate p-nitrophenyl phosphate (Jiancheng Bio, Nanjing, China). Enzyme activity was normalized against cellular protein concentration and expressed as U/g protein. Protein concentration was determined by the Bradford assay.

2.7 Von Kossa and Safranin O/Fast green staining Ligamentum flavum cells were fixed with 0.1M sodium cacodylate. Silver nitrate (3%) was added in the dark. The cells were incubated in the dark for 30 min at room temperature and then exposed to light for 1 h. Stained cells were then compared qualitatively. Cells were stained with 0.02% Fast Green (Sigma-Aldrich) for 1 min without rinsing, 1.0% acetic acid for 30 s and 1.0% Safranin O (Sigma-Aldrich) for

30 min as previously described [40].

2.8 NAD detection Levels of intracellular NAD were detected in ligamentum flavum cells using an NAD+ /NADH quantification kit (BioVision, Mountain View, CA, USA). Cells were incubated for 20 min at –70 °C and then 10 min at room temperature in NADH/NAD extraction buffer and then centrifuged at 12,000 × g for 5 min. To convert NAD to NADH, supernatant (50 µl) was mixed with NAD Cycling Mix (100 µl) and NADH Cycling Enzyme Mix (2 µl), and incubated for 5 min at room temperature. NADH developer (10 µl) was then added and the optical density was read at 450 nm every 30 min for 4 h. A standard curve of NADH was used to determine the level of NAD, which was expressed as pmol/104 cells.

2.9 Plasmids constructs To knockdown NAMPT expression, small interfering RNA constructs were generated as follows: NAMPT-shRNA (5′-TGAGTAACCTTAGGAGTCG-3′), and a scrambled negative shRNA was used as a control (5′-GACCTGTACGCCAACACAGTG-3′). The sequences were chemically synthesized (Genechem, Shanghai, China) and subcloned into a psilencer 4.1 vector (Invitrogen, NY, USA), followed by selection with puromycin (Gibco, Grand Island, NY, USA) to generate stable cell lines. For overexpression of NAMPT (NAMPT-OE), the following primers were used for amplification: forward: 5′-GCGGGTACC ATG AATCCTGCGGCAGAAGC-3′, reverse: 5′-GGCCTCGAG CTAATGATGTGCTGCTTCCA-3′. The NAMPT cDNA product was cloned into the mammalian expression pcDNA3.1(+) vector (Invitrogen) at sites KpnI and XhoI (TaKaRa, Dalian, China). Stable colonies were screened using G4182.10

Lentiviral transduction.

Full-length miR-182, miR-182-specific inhibitor, or corresponding negative control oligonucleotides were cloned into pLVX vectors (Clontech, Mountain View, CA) downstream of the green fluorescent protein (GFP) gene at XhoI and EcoRI sites. An miR-182-overexpressing lentivirus (LV-miR-182), a lentivirus that inhibits

miR-182 (LV-anti-miR-182), or a control plasmid were transfected into 293T cells along with Lenti-X HTX packaging mix (Clontech) using Lipofectamine 2000. After 48 h transfection, media containing viruses was collected. For virus titer determination, 293 T cells were seeded into 12-well plates at a density of 5 × 105 cells per well. Cells were infected with lentiviruses with a series of dilutions. Forty-eight hours after infection, the virus titer was determined by counting the number of GFP-positive cells (10 fields per well) under a fluorescence microscope. Puromycin (2 μg/ml) was added to select stably transfected cells.

2.11 Immunohistochemistry (IHC) Tissue samples were fixed in formalin and embedded in paraffin before sectioning (4-μm thickness) and deparaffinization in xylene. After dehydration in a graded series of ethanol, antigen retrieval was performed in citrate buffer for 30 min in a steamer, followed by washing in PBS. Tissue sections were then quenched in 3% hydrogen peroxide, blocked with PBS containing 10% goat serum (Sigma-Aldrich) for 1 h at 37 °C, and incubated with anti-NAMPT antibody (ab58640, 1:100 dilution), anti-RUNX2 (ab23981 1:100 dilution), anti-OCN (ab93876, 1:100 dilution), anti-OPN (ab8448, 1:100 dilution) overnight at 4°C. The signal was amplified and visualized with diaminobenzidine-chromogen, followed by counterstaining with hematoxylin.

2.12 Luciferase reporter assay The luciferase reporter assay was performed as previously described [41]. The putative miR-182 binding sites in the 3′-UTR of NAMPT were predicted using TargetScan (http://www.targetscan.org). The 3′-UTR cDNA fragments of NAMPT containing the putative wildtype or mutant miR-182 binding sites were amplified using

the

following

primers:

wild

type

5′-GCGCTCGAGTACCAGTGAAATGCCA-3′

3′-UTR

of and

NAMPT,

forward: reverse:

5′-GCCGCGGCCGCCATTTAAGTCATACCAA-3′; mutant 3′-UTR of NAMPT,

forward:

5′-GCGCTCGAGCAGTGAAATCGGTAAT-3′

and

reverse:

5′-GCC

GCGGCCGCTAGTGGAAGCAGAAAT-3′; The amplified cDNA fragments were subcloned into the psiCHECK-2 vector (Promega, USA) at the XhoI and NotI sites downstream of the luciferase gene. 293 cells were co-transfected with the luciferase reporter systems and miR-182 mimics or mimics NC as indicated in figure legends. Luciferase activity was detected 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega) following manufacturer's instructions. Data were normalized to Renilla luciferase activity.

2.12 Statistical analysis All measurements were performed on at least three independent experiments. Data are presented as mean ± S.D. Differences between groups were analyzed by Student’s t-test and one-way analysis of variance. Statistical analysis was performed using SPSS 13.0 software (SPSS, Chicago, IL, USA). Statistical significance was defined as P < 0.05.

3. Results 3.1

Histological characterization of tissues from patients with OLF Histological characterization of ligamentum flavum from patients with OLF

showed a decrease in normal fibrous alignment, increased mineralized nodule formation and the accumulation of glycosaminoglycan-rich matrix in cell cultures stained with hematoxylin, Alizarin red, Safranin O/Fast Green and Von Kossa, and in ALP assays (Fig. 1A–D). Non-OLF cells are fibroblast-like with a spindle shape. A decrease in fibrous alignment was found in OLF samples stained with hematoxylin and an increased presence of chondrocyte-like cells can be seen in cells from OLF tissue stained with Safranin O and Fast Green. Mineralized nodule formation in OLF tissue is clearly indicated by Alizarin red staining. OLF cells were positive in Von Kossa staining and ALP assays, which indicates an osteogenic phenotype and bone nodule formation.

3.2

Relative expression of miR-182, NAMPT, and osteogenic differentiation-

related proteins in OLF tissue We determined whether miR-182 and NAMPT were differentially expressed in non-OLF and OLF tissue by qRT-PCR analysis (Fig. 2A and B). MiR-182 was significantly downregulated in OLF tissue compared to non-OLF tissue whereas NAMPT was significantly upregulated. We also assessed levels of proteins related to osteogenic differentiation in non-OLF and OLF tissues by immunohistochemistry (Fig. 2C). The levels of NAMPT, RUNX2, OCN, and OPN were markedly increased in OLF tissues. This confirms the presence of osteogenic differentiation in the ligamentum flavum tissue of patients with OLF.

3.3 Overexpression of miR-182 inhibits ossification of ligamentum flavum cells First, we tested whether overexpressing miR-182 could influence the levels of osteogenic differentiation in the ligamentum flavum tissue by infecting cells with a lentivirus expressing miR-182 (LV-miR-182). mRNA and protein expression levels of NAMPT, RUNX2, OCN, and OPN in OLF cells infected with LV-miR-182 and a negative control (LV-miR-NC) were assessed by qRT-PCR and western blotting. The overexpression of miR-182 for 14 days significantly inhibited the mRNA expression and protein levels of NAMPT, RUNX2, OCN, and OPN compared to the negative control (Fig. 3A–E). Moreover, the ALP assay and Alizarin red staining revealed markedly reduced mineralized nodule formation in cells overexpressing miR-182 (Fig. 3F). These results indicate that the overexpression of miR-182 can inhibit the progress of ossification in ligamentum flavum cells.

3.4 Inhibition of ossification in ligamentum flavum cells by NAMPT knockdown and NAMPT inhibitor FK866 We then assessed the influence of silencing the expression of NAMPT in OLF cells by qRT-PCR and western blotting. Knockdown of NAMPT by NAMPT-shRNA in OLF cells resulted in a reduction of NAMPT mRNA and protein expression levels compared to a scrambled control (scr) (Fig. 4A). In addition, NAMPT knockdown also

inhibited the expression of RUNX2, OCN and OPN mRNA and protein levels (Fig. 4B–G). Moreover, ALP assay and Alizarin red staining revealed that NAMPT knockdown reduced the level of mineralized nodule formation in a similar way to miR-182 overexpression (Fig. 4H). We also used NAMPT inhibitor FK866 in OLF cells to investigate whether this would have the same effect as NAMPT knockdown. After 14 days, we assessed NAMPT, RUNX2, OCN and OPN mRNA and protein levels and the concentration of NAD+. We found that although NAMPT mRNA and protein levels were unaffected by FK866, those of RUNX2, OCN and OPN and the concentration of NAD+ were significantly inhibited in OLF cells (Fig. 5A–F). ALP assay and Alizarin red staining revealed less ossification in OLF cells treated with FK866 compared with the DMSO negative control (Fig. 5G). These results indicate that NAMPT plays a pivotal role in the ossification of ligamentum flavum cells.

3.5

Overexpression of NAMPT increases ossification in ligamentum flavum cells In contrast to NAMPT knockdown, overexpression of NAMPT promotes

osteogenic differentiation in OLF cells. We transfected OLF cells with pcDNA3.1-NAMPT (NAMPT-OE) and pcDNA3.1-NC (vector) and assessed the levels of mRNA and proteins at specified intervals for 14 days. NAMPT overexpression significantly increased mRNA and protein levels of NAMPT, RUNX2, OCN and OPN after 14 days (Fig. 6A–E), which increased the intensity of mineralized nodule formation in OLF cells (Fig. 6F).

3.6

MiR-182 targets NAMPT to inhibit the ossification of ligamentum flavum

cells To further analyze the role of miR-182 in the ossification of ligamentum flavum, OLF cells were transfected with miR-182 mimics (overexpression of miR-182), miR-182 mimics-NC (mimics negative control), miR-182 inhibitor (inhibition of miR-182) or miR-182 inhibitor-NC (inhibitor negative control) for 48 h and MiR-182 expression was detected by qRT-PCR. The expression of miR-182 was significantly

increased in cells transfected with the mimic miR-182 compared to the negative control and inhibited by the miR-182 inhibitor (Fig. 7A and B). We then determined whether the NAMPT ossification of ligamentum flavum cells involves an interaction by miR-182, we mutated the putative miR-182 binding site in NAMPT (Fig. 7C) and cotransfected 293 cells with a luciferase reporter plasmid containing wild-type or mutant NAMPT and miR-182 mimics or mimics-NC. After 48 h, luciferase activity was reduced in cells transfected with wild-type NAMPT but not in those transfected with mutated NAMPT, which suggests that miR-182 directly interacts with NAMPT (Fig. 7D). We also found that the expression of NAMPT is inhibited when miR-182 is overexpressed but this inhibition is diminished by transfection with an miR-182 inhibitor (Fig. 7E). Levels of NAMPT protein in cells overexpressing miR-182 or transfected with miR-182 inhibitor confirmed these results (Fig. 7F). To further examine the functional effects of the modulation of NAMPT expression by miR-182 in OLF cells, the levels of RUNX2, OCN and OPN mRNA and protein were analyzed by qRT-PCR and western blotting in response to co-transfection of LV-miR-182 and vector; LV-miR-182 and NAMPT-OE; LV-anti-miR-182

and

scr,

LV-anti-miR-182;

and

NAMPT

shRNA

and

LV-anti-miR-182; and FK866. The results showed that overexpression of miR-182 inhibited RUNX2, OCN and OPN mRNA and protein expression, while co-transfection with pcDNA3.1-NAMPT can suppress this effect.

When miR-182

expression is inhibited, RUNX2, OCN and OPN mRNA and protein levels are significantly increased, while co-transfection with NAMPT-shRNA or FK866 inhibits the LV-anti-miR-182 effect (Fig. 8A–D). Consequently, mineralized nodule formation in OLF cells was reduced with miR-182 overexpression, NAMPT knockdown or the addition of FK866 but increased when miR-182 was inhibited (Fig. 8E). These results indicated that miR-182 inhibits the ossification of ligamentum flavum by downregulating NAMPT. 4. Discussion The number of OLF cases has been increasing on a global scale in the last decade with incidents occurring predominately in Asian countries [42, 43].

Polymorphisms in key genes involved in osteogenesis, such as RUNX2 and COL6A1, have been implicated as one possible explanation [44, 45], although recently other factors such as spinal injury, inflammatory response, and the regulation of osteoblast differentiation have been proposed [21, 46, 47]. Yayama reported that both collagen type

II-positive

chondrocyte-like

cells

and

alkaline

phosphatase-positive

osteoblast-like cells were observed within and around the ossification fronts[48]. Zhong reported that osteocaclin and collagen type II were expressed in OLF cells, but not in non-OLF cells, indicating that osteoblast phenotype and chondrocyte phenotype were in OLF cells and may play an important role in the development of OLF. [15]. NAMPT has been implicated in the pathogenesis of inflammation and NAMPT inhibitors are thought to target inflammatory responses in spinal cord injury [49]. Emerging evidence suggests that NAMPT is involved in the regulation of RUNX2 and may serve as a potential marker for osteoblast differentiation of bone marrow-derived mesenchymal stem cells through its regulation of intracellular NAD metabolism [34, 35]. Inhibition of NAMPT by FK866 or NAMPT knockdown in bone marrow-derived mesenchymal stem cells was able to attenuate ALP activity, reduce matrix mineralization and down-regulate osteoblast-specific genes [34]. FK866 is known to bind to the interface of the NAMPT dimer and compete directly for its nicotinamide substrate [50]. Therefore, in our experiments, even though expression levels of NAMPT are unaffected by FK866, intracellular NAD levels are depleted. We investigated whether NAMPT was regulated by miR-182 in OLF cells in OLF patients. NAMPT was found to be upregulated whereas miR-182 was downregulated in OLF compared with non-OLF tissues. Li et al.[33] found that intracellular NAD concentration and NAMPT expression increase during osteogenic differentiation, which implies that NAMPT has a key role in OLF. The authors proposed that during osteogenic differentiation, stem cells enhance the activity of the NAD salvaging pathway by increasing NAMPT expression, leading to an accumulation of intracellular NAD. Increased levels of intracellular NAD promoted the activity of the NAD-dependent enzyme Sirt1 which, in turn, promotes osteogenic differentiation.

In the present study, we found that miR-182 overexpression was found to inhibit NAMPT and the osteogenesis-related proteins RUNX2, OCN and OPN. Moreover, NAMPT knockdown and inhibition also inhibited RUNX2, OCN, and OPN whereas the opposite occurred with the overexpression of NAMPT. We also mutated the putative binding site of miR-182 in NAMPT and were able to establish that osteogenesis was related to intracellular levels of NAD involving the regulation of NAMPT by miR-182. Increased levels of miR-182 expression were able to reduce mineralized nodule formation and the accumulation of glycosaminoglycan-rich matrix in OLF cells. Previous research has proposed that decreasing NAD concentration in cells with a NAMPT inhibitor leads to a dramatic increase in autophagy and that this induces cell death in C6 glioblastoma cells [51]. Inhibiting the metabolism of NAD biosynthesis has also been found to have a synergistic effect with cyclosporin-A by inducing mitochondrial and endoplasmic reticulum stress in leukemia cells while preserving normal CD34(+) progenitor cells and peripheral blood mononuclear cells [52]. Therefore, further assessment of NAD levels in response to miR-182 regulation and the association between autophagy and cell death is warranted. MiR-182 is known to promote cell apoptosis and inhibit cell viability, proliferation, invasion and migration in human osteosarcoma cells [53]. A number of studies have found that miR-182 can control cell proliferation in cancers through various mechanisms including the targeting of oncogenes [54-56]. In contrast, high expression of miR-182 has also been associated with poor prognosis such as in colorectal carcinoma [57] and in human ovarian carcinomas [58]. Further study is needed to establish whether NAD biosynthesis is a factor in these conflicting results or whether changes in differentiation due to the upregulation of miR-182 are exclusive to certain cell types and pathways. However, in this study, we did not detect the difference in NAMPT expression between OLF and non-OLF tissues by gene microarray. A higher level of type II collagen was detected in OLF tissues in a previous study, therefore, the proliferation of type II collagen is thought to result in the formation of a hypertrophied ligament

before it develops into OLF. The exact mechanism of OLF is still unclear and it is necessary for us to study this and the exact involvement of NAMPT and Collagen II expression in OLF in a future study, and we hope that our research will assist in advancing this knowledge. To conclude, we found, through dual-luciferase reporter assays, that miR-182 directly targets NAMPT and downregulates its expression in OLF cells. The overexpression of miR-182 suppressed the effects of NAMPT overexpression on upregulating RUNX2, OCN, and OPN. Overall, these data demonstrate that miR-182 has the capacity to suppress ossification of ligamentum flavum by downregulating NAMPT and thereby altering NAD biosynthesis, which led to decreased mineralized nodule formation in OLF cells. This suggests that miR-182 has potential therapeutic value in the treatment of ossification.

Acknowledgments This work was funded by the Health and Family Planning Commission of Changzhou major science and technology projects (ZD201504) and H-level Medical Talents Training Project (2016CZBJ033), and the nineteenth batch of Changzhou science and technology plan (applied basic research) (CJ20160047), and The Youth project of National Natural Science Foundation of China (81601919)

Conflicts of interest The authors have no conflict of interest to state.

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Fig. 1. Histological characterization of ossification in ligamentum flavum (OLF) tissue. (A-D) An increased presence of chondrocyte-like cells can be seen in OLF cells stained with Safranin O and Fast Green. Increased uptake of Safranin O indicates the accumulation of glycosaminoglycan-rich matrix in cell cultures. Mineralized nodule formation in OLF cells can be observed by Alizarin red staining. OLF cells stained positive with alkaline phosphatase (ALP) assay and Von Kossa indicating an osteogenic phenotype and bone nodule formation. Bar = 100 μm.

Fig. 2. Relative miR-182 and NAMPT expression in ossification of the ligamentum flavum (OLF). (A, B) The expression of miR-182 and NAMPT in non-OLF and OLF tissues used qRT-PCR analysis. (C) Immunohistochemistry of NAMPT, RUNX2, OCN and OPN proteins in non-OLF and OLF tissues. **p < 0.01. Bar = 100 μm.

Fig. 3. Overexpression of miR-182 inhibits osteogenic differentiation. (A–D) qRT-PCR was used to detect the expression of NAMPT, RUNX2, OCN and OPN in ossified ligamentum flavum (OLF) cells infected with LV-miR-182 and LV-miR-NC (negative control) for 0–14 days in osteogenic differentiation culture. (E) Western blot analysis to detect NAMPT, RUNX2, OCN and OPN protein levels in OLF cells infected with LV-miR-182 and LV-miR-NC after 14 days. (F) Alkaline phosphatase

(ALP) and Alizarin red staining were used to detect mineralized nodule formation in OLF cells infected with LV-miR-182 and LV-miR-NC for 0–14 days. Untransfected OLF cells were used as a control. * p < 0.05, **p < 0.01, and *** p < 0.001. Bar = 200 μm.

Fig. 4. Knockdown of NAMPT inhibits osteogenic differentiation. (A) Knockdown of NAMPT by NAMPT-shRNA in ossified ligamentum flavum (OLF) cells and qRT-PCR and western blots assessment of NAMPT expression. OLF cells infected with LV-miR-182 and LV-miR-NC for 0–14 days in osteogenic differentiation culture. (B) qRT-PCR analysis of NAMPT expression. (C) NAD detection. (D–F) qRT-PCR analysis of RUNX2, OCN and OPN expression. (G) Western blots analysis of RUNX2, OCN and OPN protein levels after infection with LV-miR-182 and LV-miR-NC for 14 days in osteogenic differentiation culture. (H) Alkaline phosphatase (ALP) and Alizarin red staining were used to detect mineralized nodule formation in OLF cells. Untransfected OLF cells were used as a control. * p < 0.05, **p < 0.01, and *** p < 0.001. Bar = 200 μm.

Fig. 5. NAMPT inhibitor FK866 inhibits osteogenic differentiation. Ossified ligamentum flavum (OLF) cells were transfected with 1 nM FK866 and DMSO and cultured for 0–14 days. Untransfected OLF cells were used as a control. (A–E) qRT-PCR analysis of NAMPT, RUNX2, OCN and OPN expression. (F) Western blot analysis of NAMPT, RUNX2, OCN and OPN protein levels after 14 days osteogenic differentiation culture. (G) Alkaline phosphatase (ALP) and Alizarin red staining were used to detect mineralized nodule formation in OLF cells. * p < 0.05, **p < 0.01, and *** p < 0.001. Bar = 200 μm.

Fig. 6. Overexpression of NAMPT promotes osteogenic differentiation. Ossified ligamentum flavum (OLF) cells were transfected with pcDNA3.1-NAMPT (NAMPT-OE) and pcDNA3.1-NA (empty vector) for 0–14 days in osteogenic differentiation culture. Untransfected OLF cells were used as a control. (A–D)

qRT-PCR analysis of NAMPT, RUNX2, OCN and OPN expression. (E) Western blot analysis of NAMPT, RUNX2, OCN and OPN protein levels after 14 days of differentiation. (F) Alkaline phosphatase (ALP) and Alizarin red staining were used to detect mineralized nodule formation in OLF cells. * p < 0.05, **p < 0.01, and *** p < 0.001. Bar = 200 μm.

Fig. 7. MiR-182 directly targets NAMPT. Ossified ligamentum flavum (OLF) cells were transfected with miR-182 mimics, miR-182 mimics-NC (mimic negative control), miR-182 inhibitor, or miR-182 inhibitor-NC (inhibitor negative control) for 48 h. (A, B) MiR-182 expression detected by qRT-PCR. (C) Putative miR-182 binding sites in the NAMPT 3′-UTR. (D) A luciferase reporter plasmid containing wild-type or mutant NAMPT was cotransfected into 293 cells with miR-182 mimic or negative control. Luciferase activity was determined at 48 h after transfection using the dual-luciferase assay and shown as the relative luciferase activity normalized to Renilla activity. (E, F) qRT-PCR and western blot assessment of NAMPT expression in OLF cells transfected with miR-182 mimics, mimics-NC, miR-182 inhibitor, and inhibitor-NC. **p < 0.01, and *** p < 0.001.

Fig. 8. MiR-182 inhibits osteogenic differentiation by targeting NAMPT. Ossified ligamentum flavum (OLF) cells stably expressing pcDNA3.1-NAMPT were transfected with LV-miR-182, LV-miR NC, or OLF cells stably expressing NAMPT-shRNA were infected with LV-anti-miR-182 or FK866 for 14 days in osteogenic culture. (A–D) qRT-PCR and western blot analysis of RUNX2, OCN and OPN expression. (E) Alkaline phosphatase (ALP) and Alizarin red staining were used to detect mineralized nodule formation in OLF cells. *p < 0.05, and **p < 0.01. Bar = 200 μm.