LncRNA LOXL1-AS inhibits proliferation of PDLSCs and downregulates IL-1β in periodontitis patients

Background: Oncogenic role of lncRNA LOXL1-AS has been characterized in several types of cancer. Our preliminary deep sequencing data revealed the downregulation of LOXL1-AS in periodontitis and its inverse correlation with IL-1β, a critical inammatory mediator in periodontitis. This study was therefore performed to investigate the potential interaction between LOXL1-AS and IL-1β in periodontitis. Methods: Research subjects of this study included 30 periodontitis patients and 30 healthy controls. Cell transfection, RT-qPCR, luciferase activity assay and western blots were performed in this study. Results: LOXL1-AS was downregulated in periodontitis-affected periodontal ligament stem cells (PDLSCs) than that in healthy PDLSCs. While IL-1β was upregulated in periodontitis-affected PDLSCs and was inversely correlated with LOXL1-AS. Overexpression of LOXL1-AS mediated the downregulation of IL-1β in PDLSCs. IL-1β treatment did not affect the expression of LOXL1-AS. Moreover, overexpression of LOXL1-AS led to inhibited proliferation of periodontitis-affected PDLSCs. Conclusion: This study is the rst to report the downregulation of LOXL1-AS in periodontitis. Moreover, we also propose that LOXL1-AS may inhibit the proliferation of periodontitis-affected PDLSCs and downregulate IL-1β to improve periodontitis.


Background
Periodontitis, also known as gum disease, is a common type of gum infection that is caused by bacterial infection and can damage the bone structure that supports the teeth [1]. Periodontitis is a heavy burden on public health [2]. It is estimated that severe periodontitis threat more than 11% adults worldwide and the development of periodontitis can seriously affect self-esteem, speaking ability, quality of life and nutrition conditions [3]. Gingivitis management and dietary antioxidants are the primary preventative approaches for periodontitis [4]. However, the effective treatment of periodontitis requires an intensive intervention of patients' lifestyle, such as quit smoking and alcohol abstinence [5], which is practical in many cases. Therefore, novel therapeutic treatments are urgently needed.
Previous studies on the molecular pathogenesis of periodontitis have revealed a considerable number of genetic factors involved in the occurrence, development and progression of this disease [6][7][8]. The functional characterization of the key genetic players may lead to the development of novel targeted therapies [9]. MicroRNAs (miRNAs) regulate the expression of diverse target genes and play crucial roles in the development, as well as suppression of different kinds of cancer. MiR-203 was reported to suppress the growth and stemness of breast cancer via targeting cytokine signaling 3 (SOCS3) [10].
Besides mi-RNA, the progression of many diseases including periodontitis also requires the involvement of long (> 200 nt) non-coding RNAs (lncRNAs) [11,12], which regulate gene expression to participate in diverse biological processes [13]. LncRNA LOXL1-AS is a newly identi ed critical player in thoracic aortic aneurysm [14]. And the role of LOXL1-AS in the pathogenesis of periodontitis is unknown. Our present data revealed the downregulation of LOXL1-AS in periodontitis and its inverse correlation with IL-1β, which is a critical in ammatory mediator and unexpectedly elevated in chronic periodontitis [15]. In order to extend the existing investigation on the role of LOXL1-AS and link the in ammation with periodontitis, this study was then carried out to investigate the potential effects of LOXL1-AS and IL-1β on periodontitis.

Research subjects
This study was approved by the Ethics Committee of Shanghai Yangpu Shidong Hospital. All experiments on human subjects were conducted in accordance with the Declaration of Helsinki (http://www.wma.net). Research subjects of this study included 30 periodontitis patients (18 males and 12 females, age range of 34 to 44 years old, mean age of 39.3 ± 2.1 years old) and 30 healthy controls (18 males and 12 females, age range of 33 to 44 years, mean age of 39.2 ± 2.0 years old) who were admitted at aforementioned hospital between June 2017 and June 2019. Patients' inclusion criteria: 1) newly diagnosed cases; 2) no therapies for any diseases were initiated within 3 months before this study; 3) no other systemic diseases diagnosed; 4) the patients received tooth extraction at aforementioned hospital. Exclusion criteria: 1) recurrent cases; 2) multiple chronic diseases were diagnosed. The 30 patients underwent tooth extraction due to chronic periodontitis, while the 30 healthy controls received extraction during orthodontic treatment. All patients were informed of the experimental principle of this project and signed the informed consent.

Cell culture
Periodontal ligament (PDL) tissues were extracted from one tooth of each patient and healthy controls during tooth extraction. PDL was used to make small pieces and were kept in minimal essential medium supplemented with 0.292 mg/mL glutamine, 100 µM/l ascorbic acid, 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin. Insolation of periodontal ligament stem cells (PDLSCs) was performed according to the methods described by Li et al [16].

Vectors and cell transfection
Two cases of periodontitis affected-PDLSCs with average expression of LOXL1-AS was selected to do transfection experiments. LOXL1-AS expression vector was constructed using pcDNA3.1 vector (Invitrogen) as backbone. Small interfering RNAs targeting the human IL-1β gene were designed by the Shanghai GeneChem Co., Ltd., China. The optimal sequence of small interfering RNAs against human IL-1β (5′-TCAAAGGAAAGAATCTATA-3′) was then cloned into the plasmid pGCL-GFP. IL-1β lentivirus shRNA was purchased from Shanghai GeneChem Co., Ltd., China. The negative control lentivirus shRNA was constructed by a similar process (5′-TTCTCCGAACGTGTCACGT-3′). PDLSCs were harvested at 75 -85% con uence and were counted, followed by the transfection of 10 nM vector into 10 6 cells using lipofectamine 3000 (Thermo Fisher Scienti c). Control (C) cells were untransfected cells. Negative control (NC) cells were transfected with empty pcDNA3.1 vector. The following experiments were performed using cells harvested at 48 h post-transfection.

RNA preparations and RT-qPCR
RNeasy Mini Kit (QIAGEN) was used to extract total RNAs from PDLSCs. LookOut® DNA Erase (Sigma-Aldrich) was used to digest all RNA samples to remove genomic DNAs. In cases of IL-1β treatment, PDLSCs were treated with 0, 10 and 50 ng/ml IL-1β for 48 h before the following RNA extractions. NanoDrop™ 2000c Spectrophotometer (Thermo Fisher Scienti c) was applied to measure the concentration of RNA samples. With 1000 ng total RNAs as template, RevertAid RT Reverse Transcription Kit (Thermo Fisher Scienti c) was used to perform reverse transcriptions to synthesize cDNA. With cDNA samples as template, QuantiTect SYBR Green PCR Kit (Qiagen) was used to prepare all qPCR reaction systems. The expression levels of LOXL1-AS and IL-1β mRNA were measured with GAPDH as endogenous control. All PCR reactions were repeated 3 times.

Western blots analysis
PDLSCs were harvested at 48 h post-transfection and RIPA solution (Sigma-Aldrich) was used to resuspend cell pellets containing 10 5 PDLSCs to extract total proteins. BCA assay (Sigma-Aldrich) was performed to measure protein concentrations. RNA samples were incubated in boiling water for 10 min to reach protein denaturation. Electrophoresis was then conducted using 10 % SDS-PAGE gel to separate protein molecules, followed by gel transfer to PVDF membranes. After blocking in TBST containing 5% skim milk, the membranes were incubated with rabbit primary antibodies of GAPDH (ab9845, Abcam) and IL-1β (ab9722, Abcam) at 4 °C for 18 h, followed by incubation with secondary antibody of HRP Goat Anti-Rabbit (IgG) (ab6721, Abcam) at 24 °C for 2 h. Signals were produced using ECL (Sigma-Aldrich, USA) and data were processed using Quantity One software.

Statistical analysis
Three biological replicates were included in each experiment. Mean values of 3 replicates were calculated and were used for all data analysis. Unpaired t-test was used to explore differences between two groups.
Differences among multiple groups were explored by ANOVA (one-way), followed by Tukey's post-hoc test. Correlations were analyzed by Pearson's correlation coe cient. P < 0.05 was considered as statistically signi cant.

Results
The expression of LOXL1-AS and IL-1β mRNA were altered in periodontitis-affected PDLSCs The differential expression of LOXL1-AS and IL-1β mRNA in 30 cases of periodontitis-affected PDLSCs and 30 cases of control PDLSCs was detected by qPCR. It was shown that both LOXL1-AS and IL-1β were dysregulated in periodontitis patients. Speci cally, LOXL1-AS was signi cantly downregulated in periodontitis group relative to healthy controls. (Fig. 1A,  The expression of LOXL1-AS and IL-1β were signi cantly and inversely correlated across periodontitisaffected PDLSCs Linear regression was used to analyze the correlation between the expression levels of LOXL1-AS and IL-1β mRNA across 30 cases of periodontitis-affected PDLSCs and 30 cases of control PDLSCs. It was observed that the expression levels of LOXL1-AS and IL-1β mRNA were signi cantly and inversely correlated in periodontitis group ( Fig. 2A). By contrast, there was no signi cant correlation between the expression of LOXL1-AS and IL-1β in healthy group (Fig. 2B), which suggested that the relationship between the expressions of LOXL1-AS and IL-1β was more indicatable in periodontitis condition but not in normal condition.
Overexpression of LOXL1-AS mediated the downregulation of IL-1β xdxxx Overexpression of LOXL1-AS or suppression of IL-1β led to inhibited proliferation of periodontitis-affected PDLSCs The promoted proliferation of PDLSCs in periodontitis has been demonstrated in previous literature [17]. Also, as we found the downregulated expression of LOXL1-AS in periodontitis patient, whether LOXL1-AS had any effects on the proliferation of PDLSCs was investigated. CCK-8 assay was performed to analyze the effects of overexpression of LOXL1-AS on the proliferation of 2 cases of periodontitis-affected PDLSCs. Compared to C and NC groups, overexpression of LOXL1-AS led to signi cantly inhibited cell proliferation (Fig. 4, p < 0.05). In order to investigate the effects of IL-1β on the proliferation of periodontitis-affected PDLSCs, endogenous IL-1β was knocked down (Fig. 5A, p < 0.05). Indeed, suppression of IL-1β could also inhibit the proliferation of periodontitis-affected PDLSCs (Fig. 5B, p < 0.05), suggesting that in ammation might play negative roles in the pathogenesis of periodontitis. Overall, the inhibitory effects of LOXL1-AS on the proliferation of PDLSCs with periodontitis indicates upregulation of LOXL1-AS might be an effective approach on the therapy of periodontitis.

Discussion
In this study, we investigated the function of LOXL1-AS in periodontitis. We found that LOXL1-AS was downregulated in periodontitis and might regulate the expression of IL-1β and the proliferation of PDLSCs to participate in periodontitis.
The expression pattern and function of LOXL1-AS have only been investigated in thoracic aortic aneurysm [14]. The expression of LOXL1-AS is signi cantly upregulated in thoracic aortic aneurysm and can induce aortic smooth muscle cell apoptosis and promote proliferation to participate in the pathogenesis of this disease [14]. A previous study proposed that LOXL1-AS might play different roles in regulating proliferation of different cell types [17], in which it was also shown that the abnormally increased proliferation rate of PDLSCs is involved in the development of periodontitis [17]. Our study also focused on the regulatory functions of LOXL1-AS in periodontitis condition. In this study, we rstly found that the expression levels of LOXL1-AS was decreased in periodontitis patients, which led us to infer that LOXL1-AS might be a positive and even a protective factor for periodontitis. Therefore, we overexpressed LOXL1-AS in periodontitis affected-PDLSCs to evaluate cell proliferation. Interestingly, LOXL1-AS could signi cantly inhibit periodontitis affected-PDLSCs proliferation in both two cases, indicating that LOXL1-AS is a potential positive regulator in periodontitis at least in the aspect of inhibiting cell proliferation. Therefore, upregulating the expression of LOXL1-AS might serve as a therapeutic approach for periodontitis. And inhibiting proliferation of PDLSCs by overexpressing LOXL1-AS might bene t the recovery from periodontitis.
Periodontitis is an in ammatory disease. The production of some pro-in ammatory factors, such as IL-1β, is increased in periodontitis patients to promote the in ammatory responses [18]. Consistently, our study also showed the upregulation of IL-1β in periodontitis-affected PDLSCs compared to control PDLSCs. Besides, our study reveals that upregulation of LOXL1-AS can inhibit the expression of IL-1β, which further con rm our inference that LOXL1-AS is a positive regulator for periodontitis. However, the mechanism behind this modulation is still unclear and need to be explored. In our study, it was observed that the expression levels of LOXL1-AS and IL-1β mRNA were signi cantly and negatively correlated only across periodontitis-affected PDLSCs but not control PDLSCs, which suggests that a certain pathological mediator might participate in the regulation of IL-1β by LOXL1-AS in periodontitis. Therefore, further studies are required to verify our inference. As mentioned above, the expression levels of IL-1β are increased in periodontitis patients to promote the in ammatory responses. Therefore, other two cases of periodontitis-affected PDLSCs with average expression levels of IL-1β were selected to conduct the knockdown of IL-1β and evaluate the effect of IL-1β on periodontitis-affected PDLSCs proliferation. As expected, downregulation of IL-1β could signi cantly reduce the cell proliferation rate, which suggests that in ammation might be a pessimistic factor for periodontitis, and alleviating in ammatory response would be an effective approach for periodontitis therapy. The limitations of our study are that only two cases of periodontitis-affected PDLSCs with average expression levels of LOXL1-AS were used for transfection to assess the effects of LOXL1-AS on the expression of IL-1β and cell proliferation, and more cases are needed in further research. Moreover, why IL-1β can be regulated by LOXL1-AS is still unknown, and more efforts are needed to explore the behind mechanism. Lastly, the evidence for LOXL1-AS to participate in other pathological processes of periodontitis is still required.

Conclusions
This study is the rst to report the downregulation of LOXL1-AS in periodontitis. As a positive regulator, LOXL1-AS inhibits the expression of in ammation factor IL-1β and suppresses the proliferation of PDLSCs, Accordingly, the mechanism of this research was described as the following scheme (Fig. 6).   Expression levels of LOXL1-AS and IL-1β mRNA were signi cantly and inversely correlated across periodontitis-affected PDLSCs. Linear regression was used to analyze the correlation between LOXL1-AS mRNA level and IL-1β mRNA level across 30 cases of periodontitis-affected PDLSCs (A) and 30 cases of control PDLSCs (B). The expressions of LOXL1-AS and IL-1β were negatively correlated in periodontitis patients but not healthy controls. Overexpression of LOXL1-AS mediated the downregulation of IL-1β. Two cases of periodontitis-affected PDLSCs with average LOXL1-AS expression were transfected with LOXL1-AS overexpression vector and the mRNA level of LOXL1-AS was con rmed by qPCR at 48 h post-transfection (A). The effects of LOXL1-AS elevation the expression of IL-1β at both mRNA (B) and protein (C) levels were measured by qPCR and western blots. PDLSCs were treated with 0, 10 and 50 ng/ml IL-1β for 48 h, followed by the measurement of the expression levels of LOXL1-AS, and no alteration was observed in the effect of IL-1β on LOXL1-AS expression (D). Experiments were repeated 3 times and mean values were presented. *, p < 0.05.  IL-1β knockdown resulted in the inhibited proliferation of periodontitis-affected PDLSCs. CCK-8 assay was performed to analyze the effects of IL-1β depression on the cell proliferation of periodontitis-affected PDLSCs with average IL-1β expression. Other 2 cases of PDLSCs with periodontitis was isolated and used to evaluate the effects of IL-1β knockdown on cell proliferation, and interfering endogenous IL-1β could also drive down the proliferation rate of periodontitis-affected PDLSCs. Experiments were repeated 3 times and mean values were presented. *, p < 0.05. Figure 6 The scheme of the mechanism of the effect of LOXL1-AS and IL-1β on periodontitis. In periodontitis patients, LOXL1-AS displayed an elevated level. Also, a decrease in IL-1β was observed, which contributes to the high response of in ammation in these patients. On the other hand, overexpression of LOXL1-AS was able to downregulate IL-1β expression so as to suppress in ammatory effects in PDLSCs. In addition, LOXL1-AS elevation and IL-1β reduction prohibited PDLSCs proliferation to play protective roles in the progression of periodontitis.