Knockdown of MALAT1 Inhibits the Progression of Chronic Periodontitis via Targeting miR-769-5p/HIF3A Axis

Purpose Chronic periodontitis (CP) is a long-lasting inflammatory disease that seriously affects oral health. This study is aimed at investigating the regulatory mechanism of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in CP. Methods Primary human periodontal ligament cells (PDLCs) were treated with P. gingivalis lipopolysaccharide (LPS) to establish a CP model. Quantitative real-time PCR (qRT-PCR) was used to measure the expression of MALAT1 and miR-769-5p in gingival tissues of patients with CP and LPS-treated PDLCs. Cell viability was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of inflammatory cytokines. The protein levels of caspase-3, Bax, Bcl-2, and hypoxia-inducible factor (HIF) 3A were determined by western blot assay. Dual-luciferase reporter (DLR) assay was applied to validate the target relationships between miR-769-5p and MALAT1/HIF3A. Results The expression of MALAT1 and HIF3A was enhanced, and the expression of miR-769-5p was reduced in gingival tissues of patients with CP and LPS-treated PDLCs. MALAT1 knockdown promoted cell viability and inhibited inflammation and cell apoptosis in LPS-treated PDLCs. MALAT1 targeted miR-769-5p and negatively regulated miR-769-5p expression. miR-769-5p overexpression promoted cell viability and inhibited inflammation and cell apoptosis in LPS-treated PDLCs. Besides, miR-769-5p targeted HIF3A and negatively modulated HIF3A expression. Both miR-769-5p inhibition and HIF3A overexpression reversed the inhibitory effects of MALAT1 silencing on LPS-induced PDLC injury in vitro. Conclusion MALAT1 knockdown attenuated LPS-induced PDLC injury via regulating the miR-769-5p/HIF3A axis, which may supply a new target for CP treatment.


Introduction
Periodontitis is a common inflammatory disease, which is caused by the imbalance of periodontal microbiota, such as Porphyromonas gingivalis (P. gingivalis) [1]. Chronic periodontitis (CP) is a long-lasting periodontitis disease and a chronic noncommunicable disease that destroys the integrity of the periodontium and leads to gingival swelling and bleeding, bone loss, and tooth exfoliation [2]. According to data from the World Health Organization, CP is one of the chronic noncommunicable diseases that seriously affect people's quality of life [3,4]. The current treatment methods of periodontitis including scaling, surgery, and systemic antibiotics have made great progress, but the treatment effect remains dissatisfying [5]. Thus, it is necessary to understand the molecular mechanism of CP to improve its therapy.
Long noncoding RNAs (lncRNAs) above 200 nt have been recognized to be involved in many human diseases [6]. Recently, many researches showed that lncRNAs are aberrantly expressed in periodontitis and therefore play essential roles in the development of periodontitis [7,8]. LINC00687 expression is upregulated, whereas the expression of LBX2-AS1 and LINC01566 is downregulated in periodontitis samples [9]. lncRNA PTCSC3 expression is decreased in periodontal ligament stem cells (PDLSCs) of CP patients, and its overexpression inhibits PDLSC proliferation [10]. Notably, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is also related to periodontitis progression [11]. MALAT1 expression is obviously increased in PDLSCs isolated from periodontitis patients, and its overexpression promotes cell proliferation [12]. MALAT1 is highly expressed in inflammatory gingival tissues of CP and promotes inflammatory cytokine secretion in human gingival fibroblast (HGF) cells [13]. However, the detailed molecular mechanism of MALAT1 in CP needs further study.
MicroRNAs (miRNAs) are a type of noncoding RNAs that are implicated in some pathogenic events, including periodontitis [14]. Previous studies have revealed that some miRNAs affect the occurrence and development of periodontitis, such as miR-21 [15], miR-182 [16], and miR-155-5p [17]. Additionally, miR-769-5p acts as a molecular biomarker and is involved in some immunological disorders. Chen et al. have discovered that miR-769-5p expression is upregulated in rheumatoid arthritis (RA) and gouty arthritis (GA) [18]. Besides, miR-769-5p expression is decreased in lipopolysaccharide-(LPS-) treated periodontal ligament cells (PDLCs) [19]. Previous studies suggested that the roles of miRNAs in periodontitis are regulated by lncRNAs. For example, lncRNA TUG1 mitigates cell injury and cytokine production by regulating miR-498 in LPS-treated PDLCs [20]. The inhibition effects of miR-20a on secretion of inflammatory cytokines are regulated by MALAT1 in LPStreated HGF cells [13]. However, the exact role of miR-769-5p and the interaction with MALAT1 in CP are unrevealed.
Generally, hypoxia-inducible factor (HIF) 3A is known as an oncogene in several types of human cancers, such as in ovarian [21], prostate [22], breast [23], pancreatic [24], and non-small-cell lung [25] cancers. In addition, two members (HIF1A and HIF2A) of the HIF family are confirmed to upregulate in aging gingival tissues with hypoxic stress [26]. Notably, a recent study conducted by Jia et al. has demonstrated that HIF3A expression is increased in CP tissues and LPS-induced PDLCs and HIF3A overexpression partly reversed the effects of miR-210 upregulation on cell viability, apoptosis, and inflammation factor expression in LPS-treated PDLCs [27]. Nevertheless, whether HIF3A interacts with the MALAT1/miR-769-5p axis in participation of CP progression is relatively unknown.
In our study, the expression of MALAT1, miR-769-5p, and HIF3A was measured in gingival tissues of patients with CP and LPS-treated PDLCs. Then, we investigated the effects of MALAT1 knockdown or miR-769-5p overexpression on cell viability, inflammation, and cell apoptosis in LPStreated PDLCs. Furthermore, we further explored the regulatory mechanism of MALAT1/miR-769-5p/HIF3A axis on LPS-induced PDLC injury. This study may offer an underlying target for improving treatment strategy of CP.  [28], and healthy gingival tissue samples from healthy controls were collected during crown-lengthening procedures. This study obtained the ratification of the local ethics committee, and written informed consents were acquired from all individuals.

Material and Methods
2.2. Cell Culture and Treatment. In line with previously described methods, PDLCs were isolated from healthy periodontal ligament in the middle third of the periodontal ligament root of the third molars of 5 healthy volunteers [29]. The cells were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37°C in an incubator with 5% CO 2 . PDLCs in the third generation were used in the next experiments. To establish a CP model, PDLCs were treated with 100 ng/mL P. gingivalis LPS (Sigma, St Louis, MI, USA) for 72 h.

Quantitative Real-Time Polymerase Chain Reaction
(qRT-PCR). Total RNA from gingival tissues and PDLCs was obtained by TRIzol (Invitrogen), and cDNA was produced by utilizing the PrimeScript RT Master Mix (for gene amplification, Takara) or Mir-X miRNA First-Strand Synthesis Kit (for miRNA amplification, Takara). The qRT-PCR was performed by a SYBR Green PCR Kit (Takara). Primer sequences are enumerated in Table 1. The qRT-PCR conditions were as follows: 94°C for 10 min, followed by 40 cycles at 94°C for 15 s, 60°C for 1 min, and 72°C for 1 min. The expression of MALAT1 and HIF3A was normalized by GAPDH. U6 acted as the endogenous control for miR-769-5p. Relative gene expression was measured by 2 −ΔΔCt method.

Enzyme-Linked Immunosorbent Assay (ELISA).
The supernatants of PDLCs with transfection and LPS treatment were gathered. ELISA kits (Boster, Wuhan, China) were used to measure the levels of interleukin-(IL-) 1β, IL-6, and tumor necrosis factor-(TNF-) α in supernatants according to the manufacturer's protocol. The absorbance at 450 nm was read using a microplate reader (Molecular Devices).

Target Prediction.
The miRNA targets of MALAT1 were predicted using StarBase software (http://starbase.sysu.edu .cn/), and 357 miRNA targets were predicted. Among these miRNA targets, miR-769-5p was selected due to its important role in LPS-induced periodontal ligament cells [19]. In addition, the regulatory relationship between MALAT1 and miR-769-5p has not been studied yet. The mRNA targets of miR-769-5p were predicted using StarBase software and Tar-getScan software (http://www.targetscan.org/vert_72/). A total of 2,321 and 3,669 targets were predicted, respectively. Afterwards, HIF3A was chosen due to its crucial role in periodontitis [27] and unknown relationship with miR-769-5p.
2.10. Statistical Analysis. All data were evaluated by applying SPSS 22.0 software (IBM Corp., Armonk, NY, USA) and presented as the mean ± standard deviation. The comparisons between two groups or among multiple groups were performed by Student's t-test or one-way ANOVA followed by Tukey's post hoc test. A value of P < 0:05 was deemed as a significant difference.

Inhibition of MALAT1 Attenuates LPS-Induced PDLC
Injury. MALAT1 expression was increased in gingival tissues of patients with CP compared with gingival tissues of healthy controls (P < 0:01, Figure 1(a)). As illustrated in Figure 1(b), MALAT1 expression in LPS-treated PDLCs was higher than that in control PDLCs (P < 0:01). To explore the role of MALAT1 in CP, PDLCs were transfected with si-NC or si-MALAT1. The results displayed that MALAT1 knockdown suppressed MALAT1 expression in PDLCs (P < 0:01, Figure 1(c)). MTT assay indicated that cell viability was inhibited in LPS-treated PDLCs compared with control PDLCs, while silencing of MALAT1 enhanced cell viability in LPStreated PDLCs (P < 0:01, Figure 1(d)). Moreover, the levels of IL-6, IL-1β, and TNF-α were obviously increased in LPStreated PDLCs compared with control PDLCs, while knockdown of MALAT1 inhibited the release of inflammatory cytokines in LPS-treated PDLCs (P < 0:01, Figures 1(e)-1(g)). Further studies indicated that the protein levels of Bax and caspase-3 were enhanced, whereas Bcl-2 expression was reduced in LPS-treated PDLCs compared with control PDLCs (P < 0:01, Figures 1(h) and 1(i)). Silencing of MALAT1 reduced the Bax and caspase-3 protein levels and enhanced Bcl-2 expression in LPS-treated PDLCs (P < 0:01, Figures 1(h) and 1(i)). These data revealed that silencing of MALAT1 enhanced cell viability and inhibited inflammation and apoptosis in LPS-treated PDLCs.

Gene
Sequences (5′-3′) uncovered that miR-769-5p expression in the si-MALAT1 group was increased in comparison with that in the si-NC group (P < 0:01, Figure 2(b)). Moreover, DLR assay showed that the luciferase activity of MALAT1-wt in the miR-769-5p mimics group was decreased as compared to that in the miR-NC group (P < 0:01, Figure 2(c)). These results suggested that miR-769-5p was a downstream target of MALAT1.

Discussion
CP not only seriously affects oral health but also increases the patient's risk of other chronic diseases [30,31]. Previous researches have pointed that some lncRNAs are associated with pathogenesis of periodontitis, such as lncRNA OIP5-AS1 and LINC00687 [32,33] Increasing studies showed that MALAT1 plays a pivotal role in LPS-induced inflammation models, such as acute lung injury [34] and ATDC5 cell inflammatory injury [35]. Recently, studies have demonstrated that MALAT1 participates in the development of periodontitis and is upregulated in PDLSCs [12] and inflammatory gingival tissues of CP [13]. In this study, MALAT1 expression was notably increased in  BioMed Research International gingival tissues of patients with CP and LPS-treated PDLCs, suggesting that MALAT1 was related to pathogenesis of CP. Previous studies demonstrated that MALAT1 overexpression promotes inflammatory cytokine production in LPS-treated HGF cells [13]. Similarly, our results suggested that knockdown of MALAT1 inhibited the secretion of inflammatory cytokines in LPS-treated PDLCs. Meanwhile, we found that MALAT1 knockdown enhanced cell viability and inhibited cell apoptosis in LPS-treated PDLCs. All these results suggested that MALAT1 knockdown may inhibit the occurrence and developments of CP in vitro.
Previous studies showed that MALAT1 is involved in CP pathogenesis by sponging miR-125a-3p [11] or miR-20a [13]. In this study, we found that miR-769-5p was a downstream target of MALAT1 and reversely modulated by MALAT1. Numerous studies indicated that miR-769-5p participates in the growth of some cancers and is reduced in non-small-cell lung carcinoma (NSCLC) [36] and retinoblastoma (RB) [37]. Interestingly, Du et al. have pointed that miR-769-5p expression is decreased in LPS-treated PDLCs [19]. Similarly, our results displayed that miR-769-5p expression was reduced in LPS-treated PDLCs and gingival tissues of patients with CP, suggesting that miR-769-5p may be an anti-inflammatory gene in CP. At present, the function of miR-769-5p has been explored in several types of human cancers. For example, miR-769-5p silencing can inhibit cell viability in gastric cancer cells [38]. Silencing of miR-769-5p obviously inhibits cell viability and promotes cell apoptosis in glioma cell lines [39]. However, the function of miR-769-5p is rarely discussed in inflammatory diseases. In this study, our results cleared that miR-769-5p overexpression enhanced cell viability and inhibited apoptosis in LPS-treated PDLCs. Besides, our study revealed that miR-769-5p overexpression could inhibit the secretion of inflammatory cytokines in LPS-treated PDLCs. The above results suggested that miR-769-5p makes a great deal of contributions on inhibiting the development of CP in vitro. Because MALAT1 directly targeted miR-769-5p, we speculated that MALAT1 knockdown may suppress CP progression by targeting miR-769-5p.
HIF3A, a main gene involved in the homeostatic processes, is commonly involved in chronic inflammation [26]. As a transcription factor for many target genes, HIF3A can be regulated by miR-210 in LPS-treated PDLCs [40] or modulated by miR-429 in human endothelial cells [41]. In this study, HIF3A was a target gene of and negatively regulated by miR-769-5p. Previous researches revealed that HIF3A expression is increased in periodontitis [40] and in LPS-treated BV-2 microglial cells [42]. Similar to previous results, we found that HIF3A expression was also increased in gingival tissues of patients with CP and LPS-treated PDLCs, suggesting that HIF3A may be a proinflammatory gene in CP development. In addition, Cuomo et al. have discovered that HIF3A is involved in inflammatory cell infiltration in a murine model of arteriotomy [43]. Meanwhile, HIF3A can regulate the inhibition effect of miR-210 on secretion of inflammatory factors and cell apoptosis in LPS-treated PDLCs [40]. Thus, we speculated that miR-769-5p overexpression may inhibit inflammation and apoptosis by regulating HIF3A in LPStreated PDLCs. Further studies revealed that miR-769-5p inhi-bition and HIF3A overexpression reversed the influence of MALAT1 silencing on cell viability, inflammatory factor secretion, and apoptosis-related protein levels in LPS-treated PDLCs. Because MALAT1 negatively regulated miR-769-5p and miR-769-5p negatively regulated HIF3A, we speculated that MALAT1 knockdown may alleviate LPS-induced PDLC injury by regulating the miR-769-5p/HIF3A axis.

Conclusion
In conclusion, this research revealed that the expression of MALAT1 was upregulated in gingival tissues of patients with CP and LPS-treated PDLCs. In addition, knockdown of MALAT1 enhanced cell viability and inhibited inflammation and apoptosis in LPS-treated PDLCs through regulating the miR-769-5p/HIF3A axis. This study may provide a new target for the therapy of CP.

Data Availability
All data can be obtained by contacting the corresponding author.