Cell culture
The Ethics Committee of Nanjing Medical University School of Stomatology gave clearance to this study. After obtaining the patient’s informed consent in the Department of Oral Surgery of Jiangsu Provincial Stomatological Hospital, we collected orthodontic reduction premolars from healthy donors under 16 years old. The pulp tissue was carefully isolated from immature roots, rinsed in phosphate-buffered saline (PBS; Gibco, Life Technologies, USA), sliced, and then incubated at 37 °C in a 5% CO2 incubator with 3 mg/ml collagenase type I (Sigma, St. Louis, MO, USA). The tissue block and separated cells were collected after 30 min of digestion by centrifugation at 1000 r/min for 5 min. They were inoculated at 37 °C in a 5% CO2 incubator with the complete alpha minimum essential medium (α-MEM; Gibco, Life Technologies, USA) containing 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco, Life Technologies, USA), and 10% fetal bovine serum (FBS; ScienCell Research Laboratories, California, USA). Every three days, the medium was changed. When the cells have reached 70–80% confluence, they are trypsin digested and passaged at a 1:3 ratio. Cells from stage 3 to passage 5 were utilized in the subsequent experiments.
Flow cytometry
In order to identify the phenotype of hDPSCs, flow cytometry was applied to detect MSCs’ surface markers. According to the instructions, the cells at passage 3 were incubated with anti-human CD90, CD73, CD29, CD34, and CD45 fluorescent antibodies (BD Pharmingen, San Diego, CA, USA).
Alkaline phosphatase (ALP) activity and staining
Transfected hDPSCs were planted in a 12-well plate and grown for 7 days in mineralization induction medium. The quantitative analysis of ALP activity was performed using an alkaline phosphatase detection kit (Jiancheng, Nanjing, China) in accordance with the instructions. After 30 min of fixation with 4% paraformaldehyde, ALP staining was conducted with the BCIP/NBT staining kit (Beyotime, Shanghai, China).
Alizarin red S staining (ARS) and quantification
The transfected hDPSCs grown for 14 days in mineralization induction media, with the medium changing every three days. Before being stained with Alizarin Red S (Leagene Biotechnology, Beijing, China), the cells were fixed in 4% paraformaldehyde. The mineralized nodules were seen and photographed using an inverted microscope. The staining profile was scanned using the scanner. To quantify ARS, 10 percent cetylpyridinium chloride was utilized, and the OD value was measured at 562 nm absorbance. Calcium levels were normalized to total protein levels in each group.
Stem cell multidirectional differentiation
hDPSCs were inoculated onto 6-well plates and grown in full medium to 80 percent confluence and above for odontogenic and adipogenic differentiation. 2 weeks after induction with odontogenic differentiation induction medium (10 mM β-glycerophosphate (Sigma, St. Louis, MO, USA), 50 μg/mL ascorbic acid (Sigma, St. Louis, MO, USA) and 10 nM dexamethasone (Sigma, St. Louis, MO, United States) diluted in 10% FBS α-MEM), the cells were identified with ARS. After 21–30 days of cell culture using OriCell® hDPSCs Adipogenic Differentiation Kit (Cyagen Biosciences, Guangzhou, China), Oil Red O staining was for cell identification. For chondrogenic differentiation, the right amounts of cells were cultured in a 15 ml conical centrifuge tube with OriCell® Human BMMSCs Chondrogenic Differentiation Kit (Cyagen Biosciences, Guangzhou, China) for 1–2 days to form cartilage ball. The cartilage ball was carefully inoculated into a 48-well plate for 21 days after induction, then fixed with 4% paraformaldehyde, frozen sectioned and identified by Alcian blue staining.
Quantitative reverse transcription -polymerase chain reaction (qRT-PCR)
Total cell RNA was extracted with Trizol reagent (Invitrogen, New York, NY, USA), and concentration quantification and purity detection were performed with a Nanodrop spectrophotometer. Then HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China) was used to synthesize cDNA. Finally, ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) was used for quantification. Table 1 lists the primer sequences that were employed. For miRNA, cDNA was reverse transcribed by stem-loop primers and miRNA 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). U6 was used for miRNA standardization, whereas GAPDH was utilized to normalize mRNA and lncRNA expression. The relative expression levels were determined using the 2−ΔΔCT method. In comparison with the control condition, the findings were reported as fold changes.
Cell transfection
SNHG1 overexpressed virus and specific siRNA, has-miR-328-3p mimics and inhibitor are all prepared by GenePharma (Shanghai, China) (Sequences in Additional file 1). hDPSCs were inoculated overnight and infected with lentivirus in the presence of polybrene (5 μg/mL) for 16–24 h. siRNA, mimics and inhibitors were transiently transfected with Lipofectamine 2000 (Invitrogen, New York, NY, USA) in serum-free medium for 6 h and then replaced with complete medium.
Cell counting kit-8 (CCK-8) assay
The proliferation activity of hDPSCs was detected with CCK-8 (Dojindo, Tokyo, Japan). The transfected cells were planted at a density of 2000 cells per well on a 96-well culture plate. At particular time points (day 0, 1, 3, 5 and 7), 1:9 ratios of CCK-8 reagent and α-MEM were injected into each well. OD value was determined using a microplate reader at 450 nm absorbance after 2 h of incubation.
Western blot
The total protein was extracted from the cells by protein lysate. On 10% SDS-PAGE gels, equal quantities of proteins were isolated and then transferred to 0.22 m polyvinylidene fluoride membranes (Millipore Corporation, Billerica, MA, USA). After being blocked with 5% skim milk for 1 h, the membranes were incubated with the following antibodies overnight at 4 °C: GAPDH (Proteintech, 10,494–1-AP), DSPP (Bioword, BS71212), DMP-1 (Affinity, DF8825, RRID: AB_2842022), ALP (Abcam, ab95462), Axin1 (Cell Signaling Technology, #2087), β-catenin (Cell Signaling Technology, #8480), Active β-catenin (Cell Signaling Technology, #19,807). The membranes were incubated secondary antibody for 1 h at room temperature and then exposed using the Immobilon Western Chemiluminescent HRP substrate (Millipore Corporation, Billerica, MA, USA). The protein bands were subsequently quantified with Image J software.
Immunofluorescence staining
The transfected hDPSCs were grown to an adequate density on glass coverslips in 12-well plate. The cells were rinsed in PBS and fixed with 4% paraformaldehyde. After washing with PBS, the cells were perforated with 0.25% Triton-100 (Beyotime, Shanghai, China) for 12 min and blocked with goat serum at 4 °C overnight. Then, the coverslips were incubated with DSPP and DMP-1 primary antibodies at 4 °C overnight. The coverslips were overlaid with the fluorescent dye-labeled secondary antibody and kept in dark at room temperature for 1 h. Finally, the glass coverslips were covered with the anti-fluorescence quencher agent containing 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China) and then photographed under an inverted fluorescence microscope. ImageJ software was used for semi-quantitative analysis.
Dual-Luciferase reporter assay
293 T cells were seeded to 70 percent to 80 percent confluence in a 24-well plate. Wild-type or mutant SNHG1 luciferase plasmids, as well as negative control or miR-328-3p mimics, were co-transfected into 293 T. Luciferase activity of Renilla and firefly was measured 24 h after transfection, depending on Dual-Luciferase Reporter Assay System (Promega, Madison, United States). The light intensity from firefly luciferase is normalized by Renilla luciferase.
Statistical analysis
Data from three independent experiments were analyzed by GraphPad Prism software (version 8.0; GraphPad Software, San Diego, CA) and represented the mean and standard deviation (mean ± SD). The Social Science Statistical Package (SPSS) software (version 26.0) was used for statistical analysis. To compare the differences between groups, the Student’s t test was implemented. P < 0.05 indicates statistical significance.
