hPDLSCs extraction and identification

The research was carried out following the principles of the Declaration of Helsinki and was approved by the Ethics Committee of Foshan Stomatological Hospital protocol. Signed informed consent was also obtained from all participants or guardians. Premolars extracted for orthodontic purposes between the ages of 14–18 years without dental caries or periodontal disease were used for the study. Periodontal ligament tissue was extracted from the middle of the tooth root, washed with PBS, and then subjected to digestion with 3 mg/mL collagenase I and 3 mg/mL dispose II at 37 °C for 1 h. The resulting tissue was evenly distributed in a 6 cm culture dish and maintained in a CO2 incubator at 37 °C with α-MEM medium containing 10% FBS (Gibco, USA), which was refreshed every 3 days. Following filtration through a 70 µm cell strainer, a single cell suspension was obtained, and the cells were detached with 3 mg/mL trypsin (Gibco, USA) and passaged at a 1:2 ratio when they reached 80% confluency. The third passage cells were used for subsequent experiments. Stem cell characteristics were evaluated using flow cytometry with FITC-labeled CD34, CD45, and CD90 (all from Abcam, USA).

hPDLSCs treatment and transfection

To mimic the microenvironment of PD, hPDLSCs were treated with 10 µg/ml Porphyromonas gingivalis LPS (PG-LPS, Sigma, USA) as previously reported [22]. The small interfere (si)-LSD1, miR-708-3p mimic, si-OSX and its corresponding control were obtained from Guangdong Ruibo Biotechnology Co., LTD. The hPDLSCs were cultured in osteogenic induction medium (OIM) that was replaced every 3 days.

Alizarin red staining

Following a 21-day incubation period, the cells on the plates were fixed with 2 mL of 4% paraformaldehyde per well and stained with alizarin red solution. After 30 min, the plates underwent five washes with PBS to eliminate any surplus dye. The cells were then examined, scanned, and photographed under a microscope. To determine the amount of mineralized matrix deposition, the mineral nodules were dissolved using 10% cetylpyridinium chloride (Solarbio, China), and the OD562 value was measured using a microplate reader.

ELISA

After different treatments, we gathered cell supernatants from hPDLSCs. Subsequently, the concentrations of TNF-α, IL-6, and IL-1β in these cell supernatants were determined through ELISA assays following the guidelines provided by the ELISA kit (Beyotime Biotechnology, China).

Dual-luciferase reporter gene assay

LSD1-WT and LSD1-MUT reporter vectors were generated by incorporating the binding sites of WT and MUT miR-708-3p with LSD1 fragments in the pmirGLO vector. These vectors were then transfected into 293 T cells using Lipofectamine 2000 (Qiagen, USA). 48 h later, cells were collected using a passive lysis buffer. Luciferase activity was subsequently measured using a GloMax® 20/20 luminometer (Promega, USA).

Cytoplasmic and nuclear miR-708-3p analysis

Cytoplasmic and nuclear fractionation were performed using PARIS™ Kit (Thermo Fisher, USA) following the instructions provided by the manufacturer. Briefly, hPDLSCs were collected and resuspended in pre-chilled cell fractionation buffer (200 μL), followed by centrifugation at 500 rpm for 5 min. Subsequently, RNAs were isolated, and the expression of miR-708-3p was assessed using qPCR, with U6 and GAPDH serving as reference genes.

Chromatin immunoprecipitation (ChIP) assay

The experimental procedure was performed using the chromatin immunoprecipitation kit (Millipore, USA). hPDLSCs were transfected with si-LSD1 or si-NC and subsequently cultured in OIM for 14 days. The cells were crosslinked with 1% formaldehyde for 10 min at 37 °C and then suspended in a cell lysis buffer (50 mM Tris–HCl [pH 8.1], 10 mM EDTA, 1% SDS). Following processing, the cells were lysed using a cell lysis solution, and the resulting samples were diluted tenfold with an immunoprecipitation dilution buffer. Immunoprecipitation was performed at 4 °C, and the lysate was incubated overnight with anti-LSD1 antibody (Abcam, USA), anti-H3K4me2 antibody (Abcam, USA), or nonspecific rabbit IgG. The immunocomplex was incubated with Sepharose CL-4B at 4 °C for 2 h. After multiple washes, the DNA fragments were eluted and purified, and the DNA precipitate was subjected to PCR amplification.

Quantitative PCR (qPCR)

Total RNA was extracted using TRizol (Invitrogen, USA) and reverse-transcribed into cDNA using Tiangen Biotechnology (China) kit. PCR was conducted with small RNA U6 and GADPH as internal control, using 2 × SYBR Green QPCR Master Mix from Shanghai Dongsheng Biotechnology (China). The relative gene expression was determined using the 2−ΔΔCt method. All primer sequences are provided in Table 1.

Western blotting

Protein extraction was performed using RIPA and PMSF from Shanghai Life Mode Engineering (China), and the protein concentration was determined using the BCA kit from Shanghai Dongsheng Biotechnology (China). The PVDF membrane (0.22 µm, Millipore ISEQ00010, USA) was then incubated with primary antibodies (listed in Table 2) overnight at 4 °C, followed by incubation with HRP-conjugated secondary antibodies (Abcam, USA). Protein bands were detected using Prime Western Blotting Detection Reagent (Cytiva, UK). A ChemiDoc MP imaging system (Tanon 4800, China) was used to detect chemiluminescence. Image J software was used to analyze the gray value of the bands.

Table 2 All antibodies in this studyStatistical analysis

Experimental data were analyzed using GraphPad Prism 9.0 software and statistical tests were chosen based on the distribution and variance homogeneity of the data. For normally distributed and homogeneous variance data, t tests were used to compare two groups. For multiple group comparison, either LSD analysis of variance or Dunnett’s T3 test was used based on the distribution and variance. Measurement data were presented as mean ± standard deviation, and statistical significance was determined at P < 0.05.