DPSCs culture
This study was approved by the Ethics Committee of the Affiliated Hospital of Nantong University. The cell culture and identification was performed as described in our previous studies(13). Briefly, the pulp from normal human impacted third molars was collected and digested with 3 mg/ml collagenase type I for 1 h at 37°C. Donor of the impacted third molar had given informed consent. Single-cell suspensions of dental pulp were cultured and passaged in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin at 37°C under 5 % CO2. The fourth passage cells were used in the following experiments.
Cell transfection
DPSCs were cultured in a 6-well culture plate at a concentration of 5×103/ml. After 24 h, they were transfected with KCNQ1OT1 siRNA, siRNA-NC (RuiboBio, China), overexpression vectors of KCNQ1OT1 (pcDNA-KCNQ1OT1) (Sangon Biotech, China), miR-153-3p inhibitor, inhibitor–NC, miR-153-3p mimic and mimic NC (ThemoFisher, USA) with lipofectamine 2000 (ThemoFisher, USA) according to the transfection instructions, the cells were incubated in 5% CO2, 37°C incubator and a blank control group was set up. After 48 hours, the transfected cells were taken for the following experiment.
Cell viability assay
The cell counting Kit-8 (CCK-8; Beyotime Biotechnology, China) was used to detect the viability of the DPSCs according to the manufacturer instructions, and the OD value was read at 450 nm.
Odontoblastic differentiation culture
The odontoblastic differentiation culture was performed as described in our previous study(13). Briefly, the fourth passages DPSCs were cultured in odontogenic differentiation medium containing a minimum essential medium (Invitrogen, Carlsbad, CA), 50 mg/mL a-ascorbic acid, 15% FBS, 10 mmol/L b-glycerophosphate, 10 nmol/L dexamethasone (Sigma-Aldrich, St Louis, MO), 0.292 mg/mL glutamine, 100 µg/mL streptomycin, and 100 U/mL penicillin for 14 days.
Quantitative real-time PCR (qRT-PCR)
The total RNA in cells was extracted using Trizol (Invitrogen, USA), and quantified with agarose gel (Sigma-Aldrich, USA). For LncRNA and mRNA, cDNA was synthesized using TransScript First-Strand cDNA Synthesis SuperMix (Transgen, China). For miRNA, cDNA was synthesized using TransScript miRNA First-Strand cDNA Synthesis SuperMix (Transgen, China). GAPDH and U6 were used as the internal reference. The gene expression level was calculated with 2−△△Ct method. The primer sequences were shown in the Table-1.
Luciferase reporter gene experiment
The wild-type (WT) binding fragment of lncRNA-KCNQ1OT1 or WT core sequence of 3'UTR of RUNX2 was cloned into pMIR-Report luciferase vector (Promega Corporation, USA) to construct WT-lncRNA-KCNQ1OT1 reporter vector or WT-RUNX2 reporter vector. The fragment of lncRNA-KCNQ1OT1 or core sequence of 3'UTR of RUNX2 were mutated (MU) and cloned into pMIR-Report luciferase vector to obtain MU-lncRNA-KCNQ1OT1 reporter vector or MU-RUNX2 reporter vector. The WT-lncRNA-KCNQ1OT1, MU-lncRNA-KCNQ1OT1 and miR-153-3p mimic (ThemoFisher, USA) or NC were co-transfected into HEK293 cells to investigate the relationship between lncRNA-KCNQ1OT1 and miR-153-3p. The WT-RUNX2, MU-RUNX2 and miR-153-3p mimic (ThemoFisher, USA) or NC were co-transfected into HEK293 cells to investigate the relationship between miR-153-3p and RUNX2. After transfection 48 h, relative luciferase activity was analyzed with a dual‑luciferase reporter assay system (Promega Corporation, USA) according to the manufacturer instructions.
Alkaline phosphatase (ALP) activity assay
After 14 days of odontoblastic differentiation, the cells were lysed with 1% Triton X-100 for 15 min, and centrifuged at 10,000 g for 5 min, then the supernatant was collected and detected with ALP Assay Kit (Beyotime Biotechnology, China) according to the manufacturer instructions, and the OD value was read at 405 nm.
Alizarin red staining
After 14 days of odontoblastic differentiation, the cells were incubated with 2% alizarin red staining solution (Beyotime, China) for 10 min at room temperature. Then the cells were observed under an inverted microscope and the cell mineralization was quantified with alizarin red extracted with 100 mM cetylpyridinium chloride solution (Sigma, USA), and the OD value was read at 570 nm.
Western blot analysis
After 14 days of odontoblastic differentiation, the total protein of cells was extracted by RIPA buffer (Beyotime, China). The protein was electrophoresed and transferred to PVDF membrane. After PVDF membrane blocked with 5% BSA, it was successively incubated with the primary antibody: rabbit anti-RUNX2 primary antibody (1:800, Abcam, UK), rabbit anti-DSPP (1:1000, Abcam), rabbit anti-DMP-1 (1:1000, Abcam), or mouse anti-β-actin (1:1000, Abcam, UK) and the second antibody: IRDye 700-conjugated affinity-purified goat anti-mouse (1:4,000, Rockland Immunochemicals, USA) or IRDye 800-conjugated affinity-purified goat anti-rabbit second antibody (1:4,000, Rockland Immunochemicals, USA). The relative protein expression level was analyzed with Odyssey laser scanning system (LI-COR Inc., USA).
Statistical analysis
The SPSS 22.0 software was used to analyze the data of this research. The data of this study were presented as mean ± standard deviation (M ± SD). Comparison among groups was tested with independent t test or one-way analysis of variance (ANOVA) followed by Tukey’s test. The Pearson analysis was used to analyze the correlation. Statistical graph was made using GraphPad Prism 7 software. P < 0.05 was considered statistically significant.
