The role of long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) in chronic periodontitis progression

ABSTRACT Long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) is a novel pro-inflammatory factor in severe human diseases. Since inflammatory plays important roles in periodontitis progression, we aimed to explore the role of NEAT1 in chronic periodontitis (CP) in vitro. We established a periodontitis cell model was established by Porphyromonas gingivalis lipopolysaccharide (Pg-LPS)-induced periodontal ligament cells (PDLCs). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the expression of NEAT1, microRNA (miR)-200c-3p, and tumor necrosis factor receptor-associated factor 6 (TRAF6). Cell viability, inflammatory factors, and protein expression of Bcl-2, Bax, and TRAF6 were analyzed by MTT, enzyme-linked immunosorbent assay, and Western blot. The target relationships among NEAT1, miR-200c-3p, and TRAF6 were predicted by the StarBase/TargetScan software, and further validated by dual-luciferase reporter assay. In this research, NEAT1 is up-regulated in CP tissues and periodontitis model group. Silencing of NEAT1 and over-expression of miR-200c-3p enhanced cell viability and repressed apoptosis in the periodontitis model group. NEAT1 targets miR-200c-3p, and miR-200c-3p further targets TRAF6. MiR-200c-3p inhibitor or over-expression of TRAF6 reversed the promoting effect of NEAT1 knockdown on cell viability, and the inhibiting effects on inflammatory cytokines and cell apoptosis. Consequently, the silencing of NEAT1 inhibits inflammation and apoptosis via targeting miR-200c-3p/TRAF6 axis, thereby contributing to alleviate the progression of CP. This finding could provide an underlying target for the treatment of CP.


Introduction
Chronic periodontitis (CP) is a common dental inflammatory disease [1] primarily caused by Porphyromonas gingivalis (P. gingivalis), and its virulence factor lipopolysaccharide (LPS) is the main pathogenic factor of periodontitis [2,3]. Its pathological features include increased subgingival pathogens and connective tissues injuries surrounding the teeth [4,5]. Inflammation of periodontal ligament cells (PDLCs) is important for periodontal ligament tissues regeneration [6,7]. Therefore, this study places emphasis on the regulatory mechanisms underlying the pathogenesis of periodontitis based on Pg-LPS-induced PDLCs inflammatory damage.
Long non-coding RNAs (lncRNAs) have no protein-coding ability with 200 nucleotides in length [8] and are closely associated with inflammatory-related diseases, such as CP [9,10]. Previous studies have found that over-expression of taurine-up-regulated gene 1 (TUG1) facilitates the proliferation of LPSinduced PDLCs and restrains cell apoptosis [11]. Chen et al. suggested that the up-regulation of lncRNA FGD5-antisense RNA 1 (AS1) in LPSinduced PDLCs could protect against periodontitis [12]. On the other hand, silencing of metastasisassociated lung adenocarcinoma transcript 1 (MALAT1) attenuates the inflammatory response in LPS-induced human gingival fibroblasts (HGFs) [13]. These findings imply that the abnormal expression of lncRNAs influences the progression of CP. lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) modulates inflammation in several diseases, including asthma [14], chronic obstructive pulmonary disease (COPD) [15], diabetic nephropathy [16], and sepsis [17][18][19]. Importantly, a study has reported dysregulation of lncRNA NEAT1 in gingival tissues or blood samples of patients with periodontitis in comparison with healthy subjects [20]. Huang et al. indicated that the level of lncRNA NEAT1 was up-regulated in PDLCs subjected to compressive force by qRT-PCR and RNA sequencing assay [21]. However, studies of CP involving NEAT1 are restricted to the level of tissues. Therefore, the mechanism of NEAT1 involved in CP in cellular level needs to be further elucidated.
Tumor necrosis factor receptor-associated factor (TRAF) is an oncogene in the pathogenesis of several human cancers, including colorectal [26], gastric [27], breast [28], and prostate [29] cancers. In recent years, the pro-inflammatory role of TRAF6 has attracted increasing attention [30,31]. TRAF6 is regulated by lncRNA MIAT aggravates the inflammatory response in LPS-induced septic cardiomyopathy [30]. Silencing of TRAF6 has a nephroprotective effect on LPS-induced acute renal injury by suppressing inflammation [31]. P22077 could inhibit inflammation and reduce the lung injury by promoting TRAF6 degradation in LPS-induced endotoxemia mice [32]. The down-regulation of TRAF6 has a suppressive effect on inflammation in PDLCs induced by P. gingivalis [33]. Furthermore, TRAF6 is also negatively regulated by miR-146a in CP [22]. Nevertheless, the interactions among TRAF6, NEAT1, and miR-200c-3p axis in the pathogenesis of CP are relatively unknown.
In this research, we employed CP tissues and LPS-induced PDLCs in vitro to determine whether NEAT1 is involved in the regulation of CP and what is the underlying mechanism in the progression of CP. The result showed that NEAT1 was up-regulated in CP tissues and model group. The silencing of NEAT1 could protect the PDLCs against LPS-induced inflammation and apoptosis by targeting miR-200c-3p/TRAF6 axis, thereby contributing to alleviate the progression of CP. The results may provide a novel insight for the pathophysiology mechanism of CP and may provide support for NEAT1 in the clinical applications of CP therapy.

Tissues collection
In total, 28 patients with CP without other diseases were selected from 2017 to 2018 in our hospital. Simultaneously, 20 healthy volunteers undergoing a physical examination were recruited. Gingiva tissues were obtained through operation from CP patients, followed by an original ineffectual nonsurgical scaling and root planning in accordance with the established professional and required oral hygiene of patients. Gingival tissues were also procured through crown-lengthening procedures from healthy individuals with the following inclusion criteria: clinical attachment loss <4 mm, probing depth (PD) <4 mm, and no alveolar bone destruction at radiographic level [12]. All the collected gingival tissue samples were frozen in liquid nitrogen and then stored immediately at −80°C for further experiments. The procedures were conducted based on the Declaration of Helsinki and obtained the approval of our hospital's ethics committee. Each participant has obtained informed consent.

Isolation, culture, and transfection of human PDLCs
PDLCs were isolated from the healthy periodontal ligament in the middle third of the periodontal ligament root of the third molars as previously described [34]. The cells were cultured in Dulbecco's Modified of Eagle's Medium (DMEM). Immunohistochemistry and Alizarin red staining were used to identify the PDLCs as previously described [34]. Immunocytochemistry showed that cells with a spindle shape were positive in vimentin and negative in keratin. Alizarin red staining revealed that the cells could osteogenically differentiate. Consequently, these cells were identified as PDLCs. PDLCs in the third generation were used in the next experiments [35]. PDLCs were then divided into a control group and a model group. PDLCs induced by Pg-LPS (10 μg/mL) were served as the periodontitis model group [36]. These cells served as the control (without LPS treatment). shRNAnegative control (sh-NC), sh-NEAT1, miR-200c-3p inhibitor, miR-200c-3p mimics, miR-NC, and overexpression TRAF6 vector (pcDNA-TRAF6) were cotransfected with LPS-induced PDLCs for 48 h. PDLCs were collected to perform further experiments.

Quantitative real-time PCR qRT-PCR
The total RNA from PDLCs was extracted using RNAiso Plus (Takara, Tokyo, Japan). RNA was reversed transcribed into cDNA using M-MLV Reverse Transcriptase kit (Sigma-Aldrich). qRT-PCR was performed using a SYBR® Green PCR Kit (Sigma-Aldrich) according to the instructions of the manufacturer. GAPDH or U6 was used for the normalization of the mRNA expression of NEAT1, TRAF6, and miR-200c-3p.

Statistical analysis
Data were expressed as the mean ± SD and analyzed using SPSS 20.0 (SPSS; Chicago, USA). Student's t-test was used to analyze the differences between the two groups. One-way ANOVA followed by Tukey posthoc tests and two-way ANOVA was used for comparisons between two groups or among multiple groups. P< 0.05 was considered to be a statistically significant difference.

Results
In this study, we established a periodontitis cell model by Pg-LPS-induced PDLCs. We determined the levels of TNF-α, IL-1β, and IL-6, as well as protein expression of Bcl-2, Bax, and TLR4 by ELISA and Western blot. The expression of NEAT1, miR-200c-3p, and TRAF6 was detected by qRT-PCR. The results showed that the silencing of NEAT1 could protect the PDLCs against LPSinduced inflammation and apoptosis by targeting miR-200c-3p/TRAF6 axis, thereby contributing to alleviate the progression of CP.

Knockdown of NEAT1 inhibits inflammatory responses in model group
The expression of NEAT1 was initially determined by qRT-PCR, and we found that NEAT1 was highly expressed in CP tissues (Figure 1a, p < 0.01) and the model group compared with respective controls (Figure 1b, p < 0.01). qRT-PCR assay showed that the expression of NEAT1 was decreased in sh-NEAT1 group compared with sh-NC group (Figure 1c, p < 0.01), suggesting the successful transfection of sh-NEAT1. The viability of PDLCs was decreased after LPS stimulation, while sh-NEAT1 reversed the inhibitory effect of LPS on cell viability (Figure 1d, p < 0.01). Thereafter, the influences of NEAT1 silencing on inflammatory responses were researched. As illustrated in Figure 1e-g, the levels of IL-6, IL-1β, and TNF-α were distinctly promoted in model groups. However, the promoting roles of LPS in these inflammatory cytokines were reversed by sh-NEAT1 (P < 0.01). Western blotting assay uncovered that the LPS treatment had a promoting effect on Bax level, and a suppressive effect on Bcl-2 protein expression (Figure 1h, p < 0.01). As expected, the transfection of sh-NEAT1 significantly reversed the effects of LPS on Bax and Bcl-2 (Figure 1h, p < 0.01). LPS was also found to dramatically elevate the Bax/Bcl-2 ratio, which was reversed by the transfection of sh-NEAT1 (Figure 1i, p < 0.01).

Over-expression of miR-200c-3p represses inflammation of in model group
In order to explore the function of miR-200c-3p in CP, the expression of miR-200c-3p was detected by qRT-PCR, and we found that miR-200c-3p was down-expressed in CP tissues and model group (Figure 3a-b, P < 0.01). Then, miR-200c-3p mimics were successfully transfected into model group to explore the function of miR-200c-3p on CP progression in vitro (Figure 3c, p < 0.01). As presented in Figure 3d-g, miR-200c-3p mimics remarkably facilitated the viability (P < 0.01) and repressed the levels of IL-6, IL-1β, and TNF-α (P< 0.01). In addition, miR-200c-3p mimics downregulated the expression of Bax and up-regulated Bcl-2 expression, as well as decreased Bax/Bcl-2 ratio (Figure 3h-i, P < 0.01).

TRAF6 is a target of miR-200c-3p
The binding site between miR-200c-3p and TRAF6 was predicted using TargetScan software, and a binding site is shown in Figure 4a. Western blot assay showed that TRAF6 expression was significantly decreased by the transfection of miR-200c-3p mimics (Figure 4b, p< 0.01). The DLR assay indicated that luciferase activity significantly decreased in PDLCs co-transfected with TRAF6-wt and miR-200c-3p mimics by contrast to in the TRAF6-wt + miR-NC group (Figure 4c, p< 0.01).

Silencing of NEAT1 inhibits inflammation and apoptosis by targeting miR-200c-3p/TRAF6 axis
The expression of TRAF6 in CP tissues and model group was visibly up-regulated compared with respective controls (Figure 5a-b, P < 0.01). At the same time, we analyzed the protein level of TRAF6 in model group. The results showed that protein expression of TRAF6 was downregulated by sh-NEAT1, while miR-200c-3p inhibitor partly reversed the effect of sh-NEAT1 on protein expression of TRAF6 in model group (Figure 5c, p < 0.01). Subsequently, feedback verification experiments were performed to investigate the interactions among NEAT1, miR-200c-3p, and TRAF6 on CP progression in vitro. As illustrated in Figure 5d, the transfection of miR-200-3p inhibitor and pcDNA-TRAF6 reversed the promoting effect of sh-NEAT1 on viability in model groups (Figure 5d, p < 0.01). The anti-inflammatory effects of sh-NEATT were markedly reversed by the transfection of miR-200-3p inhibitor and pcDNA-TRAF6 in model groups (Figure 5e-g, P < 0.01). Moreover, miR- 200-3p inhibitor and pcDNA-TRAF6 also reversed the effects of sh-NEATT on the expression of Bcl-2 and Bax, and the ratio of Bax/Bcl-2 in model groups (Figure 5h-i, P < 0.01).

Discussion
A study showed that Pg-LPS inhibited cell viability and triggered inflammation of PDLCs [36]. Consistent with this study, our result showed that LPS stimulation reduced the viability of PDLCs and increased the levels of inflammatory factors (IL-6, IL-1β, and TNF-α) in model groups.
Furthermore, LncRNA NEAT1 plays a crucial role in several inflammation diseases [14,16,18,37]. The up-regulation of NEAT1 occurs in the tissues of patients with asthma, COPD, and acute kidney injury [14,16,38]. In this study, we found that NEAT1 was significantly up-expressed in CP tissues. Consistent with our results, Sayad et al. also indicated that NEAT1 expression in CP tissues is dramatically elevated [20]. Additionally, we showed that the expression of NEAT1 was upregulated in model group, suggesting that NEAT1 may play a vital role in CP. To further explore the exact role of NEAT1 in CP, relevant in vitro experiments were performed in model groups. We demonstrated that the suppression of NEAT1 facilitates viability and inhibits apoptosis and inflammation in model group. In line with these results, Yi et al. discovered that knockdown of NEAT1 could promote cell viability, and suppress inflammatory factors and cell apoptosis in sepsisinduced acute kidney injury [38]. Therefore, we speculated that the silencing of NEAT1 might be a suppressor in the occurrence and development of CP. miRNAs act as suppressors to participate in the inflammation reaction of CP in vitro or in vivo [23,39,40]. A decreased expression of miR-335-5p has been found in periodontitis tissues [40], and miR-210 is low-expressed not only in CP tissues but also in model group [23]. Additionally, miR-218 is minimally expressed both in CP tissues and periodontal ligament progenitor cells [39]. In this study, miR-200c-3p was reduced in CP tissues and PDLCs, and over-expression of miR-200c-3p reduced the levels of inflammatory factors in model group, which was consistent with previous studies [24,25]. Additionally, in the present study, we demonstrated that the up-regulation of miR-200c-3p promoted cell viability and inhibited apoptosis in model group. More importantly, miR-200c-3p was found to be up-regulated by sh-NEAT1, and a target of NEAT1. Our feedback verification experiments suggested that inhibition of miR-200c-3p reversed the effects of sh-NEAT1 on cell viability, cell apoptosis, and inflammatory factors in PDLCs. The above data indicated that silencing of NEAT1 inhibited apoptosis and inflammation model groups by up-regulating miR-200c-3p expression, thereby contributing to alleviate the progression of CP.
TRAF6 is involved in the regulation of CP [33,41] and is highly expressed in LPS-induced PDLCs [41]. LPS stimulation elevates TRAF6 expression in PDLCs [33]. Similarly, we found that the protein level of TRAF6 was up-regulated in the model group, and an increased expression of TRAF6 in CP tissues was observed. The result indicated that TRAF6 may be closely related to the progression of CP. Simultaneously, we verified that TRAF6 was a target gene of miR-200c-3p and silencing of NEAT1 down-regulated TRAF6 expression. The results of the feedback verification experiment verified that the over-expression of TRAF6 reversed the anti-inflammation and antiapoptosis effects, as well as the promoting effect on the cell viability of sh-NEAT1. In other words, we believed that silencing NEAT1 alleviated the progression of CP via regulation of the miR-200c-3p/TRAF6 axis.

Conclusions
In conclusion, NEAT1 was significantly highly expressed in CP tissues and LPS-induced PDLCs. Silencing of NEAT1 markedly suppressed the  inflammatory response and apoptosis via the miR-200c-3p/TRAF6 axis in the model group. Accordingly, NEAT may act as a potential therapeutic target for CP therapy in clinical applications. In addition, we failed to verify the NEAT1/ miR-200c-3p/TRAF6 axis in vivo, and this may be a limitation of this study. We will elucidate this in the future.

Highlights
(1) NEAT1 is up-regulated in CP tissues and model group.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported that there is no funding associated with the work featured in this article.