LncRNA NEAT1 promotes nucleus pulposus cell matrix degradation through regulating Nrf2/ARE axis

Background This study aimed to assess the role and mechanism of lncRNA NEAT1 in intervertebral disc degeneration (IVD). Methods LncRNA profile (GSE56081) between IVD and healthy control was downloaded from the Gene Expression Omnibus (GEO) database and analyzes differential lncRNA expression. Expression of lncRNA NEAT1 in IVD tissue and TNF-α/IL-1β-stimulated nucleus pulposus cells were further measured by RT-PCR. The lncRNA NEAT1 overexpression plasmids (pcDNA-NEAT1) were constructed and transfected into nucleus pulposus cells. Catabolic biomarkers (MMP-3 and MMP-13), anabolic biomarkers (Col II and Aggrecan) and Nrf2 expression were further measured. To further investigate the function of Nrf2, nucleus pulposus cells were pretreated with or without 25 μM tert-Butylhydroquinone (TBHQ), a Nrf2 activator, for 18 h and subsequently cotreated with pcDNA-NEAT1. Results A total of 1432 lncRNAs were differentially expressed in GSE56081. Bioinformatic analysis found that these lncRNAs mainly enriched in Nrf2/ARE signaling pathway. LncRNA NEAT1 was highly expressed in IVD tissues than that of healthy control. Moreover, TNF-α/IL-1β induced a time- and dose-dependent increase in the mRNA expression of lncRNA NEAT1 in the nucleus pulposus cells. Overexpression of lncRNA NEAT1 abates promotes nucleus pulposus cells proliferation but induces matrix degradation. Meanwhile, nucleus and cytoplasm Nrf2 expression was significantly down-regulated by lncRNA NEAT1 upregulation. Nrf2 activator (TBHQ) could partially reverse the inhibitory effects of overexpression of lncRNA NEAT1 on matrix degradation. Conclusion Collectively, our data unveiled the lncRNA NEAT1 promotes matrix degradation by regulating Nrf2/ARE signaling pathway, suggesting a potential therapeutic for IVD in the future.


Introduction
Intervertebral disc degeneration (IVD) is mainly responsible for back pain and is a significant source of morbidity in our society [1,2]. The risk factors for IVD comprise overload, obesity, genetic variants, and nucleus pulposus cell aberrant proliferation and finally resulted in increasing of inflammatory cytokines and matrix degradation [3,4]. Treatment options for IVD include symptomatic management and/or surgery treatment [5,6]. However, these treatments can only alleviate the symptoms, but cannot fundamentally improve the condition [7]. The pathogenesis of IVD is complicated and has not yet been fully elucidated. Thus, it is necessary to explore the molecular mechanism and pathogenesis of IVD and to search for therapeutic molecular targets.  26:11 Long intergenic noncoding RNA (LncRNA) is a growing family of transcripts of length > 200 nt that have a low protein-coding potential [8]. Accumulating evidences have shown that LncRNA play a vital role in cell proliferation, metabolism and apoptosis [9][10][11]. Meanwhile, lncRNAs are aberrantly expressed in IVD and thus speculated in the progress of IVD. For example, Chen et al. [12] reported that LINC01121 act as a sponge of miR-150-5p to induced IVD progression. Cui et al. [13] found that LncRNA MAGI2-AS3 is down-regulated in IVD, which was correlated with expression of FasL. LncRNA nuclear enriched abundant transcript 1 (NEAT1), a new lncRNA, has been demonstrated to serve an important role in cell apoptosis [14], non-small cell lung cancer invasion [15] and sepsis progression [16]. However, underlying mechanisms of NEAT1-regulating functions in IVD have yet to be elucidated.
The Nrf2/ARE signaling pathway have been shown to be involved in the regulation of cell proliferation, differentiation, migration and apoptosis [17,18]. Nrf2 was modulated by PI3K/Akt signaling pathway which also played a crucial role in cell proliferation and apoptosis. Previous study revealed that Nrf2/ARE pathway was abnormally inactivated and thus speculated Nrf2/ARE signaling pathway may be used a treatment target for IVD degeneration [19]. However, whether lncRNA NEAT1 regulates nucleus pulposus cell proliferation through Nrf2/ARE signaling pathway was unknown.
In this study, we investigated differentially expressed lncRNAs between human nucleus pulposus (five degenerative as IVD and five normal specimens as control) from GSE56081. First, we proved that lncRNA NEAT1 was up-regulated in the degenerative nucleus pulposus (NP) tissues than normal specimens. Second, lncRNA NEAT1 overexpression induced cell growth and regulated extracellular matrix (ECM) expression in NP cell through regulation of Nrf/ARE pathway.

Differential expression analysis
GSE56081 was downloaded from the NCBI GEO database (https ://www.ncbi.nlm.nih.gov/geo/). In total, five degenerative samples and five normal specimens were used in this GEO dataset. Sequencing was performed on Arraystar Human LncRNA microarray V2.0 (Agi-lent_033010 Probe Name version) platform. The lncRNA expression profiles were first normalized by the Z-score and then subjected into analysis using this R package [20]. We identified differentially expressed lncRNAs with the limma package. Data visualization (heatmap and volcano plot) was performed using ggplot2 package [21]. KEGG pathway analysis was obtained from the R packages (R package, version 2.5.0).

LncRNA NEAT1 subcellular localization
The subcellular localization of LncRNA NEAT1 in cells was identified with bioinformatics tools (http://lncat las. crg.eu/) [22]. LncRNA NEAT1 subcellular localization in nucleus pulposus cells was detected by use of a FISH kit (Roche Applied Science, Germany) and nucleus was stained using DAPI (Solarbio, Beijing, China).

Patients and samples
Normal intervertebral disc tissues (n = 20) were acquired from idiopathic scoliosis patients and degenerative intervertebral disc tissues (n = 60) were obtained from IVD patients that preparing for discectomy. Pfirrmann grades according to T2-weighted section images were used to assess the degree of IVD. According to Pfirrmann grading, grades I and II indicate normal discs, whereas grades III, IV, and V indicate degenerated discs [23].

Cell culture
Human nucleus pulposus cells were isolated from normal intervertebral disc tissues of patients as described previously. In brief, intervertebral disc tissues were obtained and kept in the culture medium. Then, nucleus pulposus tissue was separated and cut into small pieces. Fragments were then digested in 0.2% type II collagenase at 37 °C for 4 h. Then, isolated cells were cultured in Dulbecco's modified Eagle medium containing 20% fetal bovine serum (FBS, Gibco, Waltham, MA) in a 37 °C 5% CO 2 incubator. The third generation of nucleus pulposus cells was used for next experiments. Different concentrations of tumor necrosis factor-alpha (TNF-α, 0, 10, 25 and 50 ng/ml) and interleukin-1β (IL-1β, 0, 5, 10 and 20 ng/ml) were used to induce degeneration of nucleus pulposus cells. Nucleus pulposus cells were grown to confluence and stimulated with TNF-α (50 ng/ml) or IL-1β (20 ng/ml) for 0, 24, 48 and 72 h.

Cell transfection
The pcDNA-NEAT1 plasmid and pcDNA empty vector were constructed and purchased from Shanghai GeneChem Co., Ltd. Nucleus pulposus cells (1 × 10 5 ) were seeded in a 6-well plate and then transfected with pcDNA-NEAT1 and pcDNA at a final concentration of 5 mg/l using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). After 24 h of transfection, the transfection efficiency was verified by qRT-PCR and the subsequent experimental procedure was performed. To further explore the molecular mechanism of lncRNA NEAT1 actions on nucleus pulposus cell proliferation, we activated the Nrf2 expression by tert-Butylhydroquinone (TBHQ) (Nrf2 activator,  Elmer) to quantify the exposed films. The relative level of total protein was normalized to GAPDH and the relative level of nuclear protein was normalized to Lamin.

Statistical analysis
Three independent repetitions were performed for all experiments. Data were expressed as mean ± SD. Oneway analysis of variance (ANOVA) followed by a Dunnett's t test was performed for three and more groups.
A p value less than 0.05 was considered to indicate statistical significance. Statistical analyses were performed by SPSS 20.0 (SPSS, Inc., Chicago, IL, USA).

Bioinformatic analysis of GSE56081
GSE56081, which including five degenerative samples and five normal samples, was used to identify the differentially expressed lncRNA. A total of 1432 lncRNAs were differentially expressed, among which 965 were up-regulated and 467 lncRNA were down-regulated (Fig. 1a, b). We selected lncRNA NEAT1 for further research mainly based on its expression was most statistically significantly. Top canonical pathways included Nrf/ARE signaling pathway, pertussis, PPAR signaling pathway, change disease, vitamin digestion and absorption, prion disease, systemic lupus erythematosus and fat digestion and absorption (Fig. 1c). LncRNA NEAT1 was mainly located in the nucleus, which were predicted by lncATLAS website (Fig. 1d) and further verified by FISH (Fig. 1e). Therefore, lncRNA NEAT1 can play a regulatory role as downstream gene expression.

LncRNA NEAT1 has high expression in IVD tissue and cell models
To detect the gene expression of lncRNA NEAT1, we used real-time PCR to detect lncRNA NEAT1 mRNA expression levels in IVD tissue and TNF-α/IL-1βstimulated nucleus pulposus cells at different time points. LncRNA NEAT1 expression level increased with the severity of the IVD increased (Pfirrmann grades: II-IV, Fig. 2a). Moreover, the expression of lncRNA NEAT1 was also significantly increased in IVD tissue when compared with normal tissues (Fig. 2b). TNF-α and IL-1β were two common pro-inflammatory cytokines that used to establish the IVD cell model. With the increase in TNF-α or IL-1β concentration and duration, the lncRNA NEAT1 expression gradually increased with statistical significance (Fig. 2c-f ).

LncRNA NEAT1 inhibits Nrf2 to promote nucleus pulposus cell matrix degradation
The expression levels of Nrf2 in the nucleus and cytoplasm were assessed by Western blot assay. The Western blot results showed that overexpression of lncRNA NEAT1 inhibited the expression of Nrf2 in nucleus and cytoplasm in nucleus pulposus cells (Fig. 4). To further evaluate the role of the Nrf2 signaling pathway in nucleus pulposus cell degradation, the Nrf2 signaling pathway activator TBHQ was used to investigate the effects of overexpression of lncRNA NEAT1 on IVD mediated by Nrf2 signaling. The Nrf2 expression increased significantly after pretreatment with Nrf2 activator (TBHQ, Fig. 5a). Nucleus pulposus cell proliferation induced by overexpression of lncRNA NEAT1 is partially blocked by TBHQ (Fig. 5b).
These results were further validated by Western blot assay (Fig. 5d).

Discussion
This study investigated the role and mechanism of lncRNA NEAT1 in occurrence and development of IVD. First, we searched GEO database and found that lncRNA NEAT1 was high expressed in the IVD tissue than normal tissue. LncRNA NEAT1 expression was up-regulated by IL-1β-and TNF-α-stimulated nucleus pulposus cells. Our results suggested that lncRNA NEAT1 expression was positively correlated with degree of degeneration grade. Overexpression of lncRNA NEAT1 significantly promotes the matrix degradation, which mainly through target with Nrf signaling pathway. LncRNA NEAT1 plays an important role in many diseases, such as gastric cancer [24], prostate cancer [25], renal fibrosis [26], and osteosarcoma [27]. What is more, Liu et al. [28] found that knockdown of lncRNA NEAT1 inhibit SK-N-SH cells apoptosis, inflammation and cytotoxicity. We found that lncRNA NEAT1 was differentially expressed in IVD tissue and IL-1β/TNF-α stimulated nucleus pulposus cells. Thus, we speculated that lncRNA NEAT1 may affect the disc degeneration.
The functional roles of lncRNA NEAT1 in cells was analyzed by loss function assays. Overexpression of lncRNA NEAT1 significantly promotes the degradation of matrix. Moreover, lncRNA NEAT1 significantly downregulated the Nrf2 expression in nucleus and cytoplasm. Those results suggested that lncRNA NEAT1 may target Nrf2 expression to regulate the matrix degradation.
Activation of the Nrf2 signaling pathway helps protect cells from oxidative stress [29,30]. Oxidative stress could enhance the Nrf2 activity, and upon activation, it would translocate into the nucleus [18]. Tang et al. [31] found that a Nrf2 gene knockout leads to IVD degeneration, which mainly through regulation of autophagy in mouse model. Overexpression of lncRNA NEAT1 inactivated the Nrf/ARE signaling pathway. This result was corroborated by analyzing the expression of Nrf2 protein in the nucleus and cytoplasm. To explore this further, Nrf2 activator (TBHQ) was applied to determine whether lncRNA NEAT1 inactivated the Nrf2 signaling pathway to induce matrix degradation. Pretreatment with TBHQ following pcDNA-NEAT1 significantly increases anabolic biomarkers (Col II and Aggrecan) and decreases catabolic biomarkers (MMP-3 and MMP-13). These results suggested that lncRNA NEAT1 promotes matrix degradation through regulation Nrf/ARE signaling pathway.

Conclusion
In short, we first elaborate the role and mechanism of lncRNA NEAT1 in IVD progression. We suggested that the lncRNA NEAT1 expression was up-regulated in degenerated NP tissues. Overexpression of lncRNA NEAT1 induced matrix degradation via modulating Nrf2/ARE signaling pathway. Our data proved that lncRNA NEAT1 may be useful as a new potential therapeutic target in IVD.