Cell culture
The cell culture process was performed as described in our previous studies [6, 20] and approved by the Ethics Committee of the Affiliated Hospital of Nantong University. Briefly, normal human impacted third molars were collected from patients of 14~22 years of age (n = 10) after they had given informed consent. The pulp was digested in a solution of 3 mg/mL collagenase type I for 1 h at 37 °C. Single-cell suspensions of dental pulp were seeded into 25-cm2 culture dishes and cultured in Dulbecco’s 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. Cells were passaged at the ratio of 1:3 when they reached 85–90% confluence. The cell populations were characterized by positive staining with anti-CD34, STRO-1, and c-kit, and by the absence of CD45. Cells from the fourth passage were used in all experiments.
Odontoblastic differentiation
The odontoblastic differentiation process was performed as described in our previous study [6]. Briefly, DPSCs from the fourth passages were cultured in odontogenic differentiation medium containing a Minimum Essential Medium (Invitrogen, Carlsbad, CA), 15% FBS, 10 mmol/L β-glycerophosphate, 50 mg/mL α-ascorbic acid, 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.
The expression of miR-140-5p was detected by qRT-PCR
The RNA in cells was extracted using TaqMan miRNA isolation kit, then the expression of mature miR-140-5p was detected by the TaqMan miRNA assay and TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA). U6 was used as the internal reference. The qRT-PCR relative quantitative method was used to analyze the experimental result.
Cell transfection
The miR-140-5p mimic (abm, Canada), miR-140-5p inhibitor (abm, Canada), corresponding negative control, restructuring plasmid pcDNA3.1-Wnt1, and empty vector control pcDNA3.1 (GenePharma, Shanghai, China) were transfected into DPSCs using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction.
Alizarin red S staining
The staining process was performed as described in our previous study [21]. The differentiated cells were fixed with 4% paraformaldehyde (PFA) for 30 min and washed with PBS. Subsequently, cells were incubated with 2% Alizarin red S solution for 10 min at room temperature under gentle agitation. Cells were then washed with deionized water to remove excess staining. Mineralization was quantified by extracting the Alizarin red S stain with 100 mM cetylpyridinium chloride solution (Sigma-Aldrich) at room temperature. The absorbance of the extracted Alizarin red S stain was measured at 570 nm.
Cell activity assay
DPSC activity was measured by using methyl-thiazole-tetrazolium (MTT) assay as described by Peng et al. [22]. MTT (AMRESCO) solution (5 mg/mL) was added to the cultures (10 μL MTT solution per 100 μL medium), followed by the incubation with 5% CO2 at 37 °C for 4 h. A solution of sodium dodecyl sulfate (SDS) was added to the cultures, which was incubated for 20 h at 37 °C in 5% CO2. The optical density (OD) was read on a Universal Microplate Reader (Synergy 2, Bio-Tek Instruments, Inc., USA) using a test wavelength of 570 nm.
Cell proliferation assay
DPSC proliferation was measured by using Ki67 immunofluorescence. Cells were first fixed in 4% PFA, followed by incubation in hydrogen peroxide, and then incubated with the rat anti-Ki67 antibody (1:800, Abcam, Cambridge, UK) overnight. The following day, the cells were labeled the secondary antibody with Alexa Fluor 568-conjugated goat anti-rat (1:1000, Molecular Probes). The cell nuclei were counter-stained with Hoechst33342 for 30 min. Positive cells were observed under a fluorescent microscope.
Western blot analysis
The cell total protein analysis process was performed as described in our previous study [20]. Briefly, the separated proteins were transferred onto polyvinylidene difluoride membranes, then the membranes were blocked with 5% nonfat milk and incubated with primary antibodies and second antibodies in turn. The following primary antibodies were used: rabbit anti-Wnt1(1:1000, Abcam), rabbit anti-β-catenin (1:1000, Abcam), rabbit anti-dentin sialophosphoprotein (DSPP) (1:1000, Abcam), rabbit anti-dentin matrix protein-1 (DMP-1) (1:1000, Abcam), and mouse anti-β-actin (1:1000, Santa Cruz). The second antibodies were goat-anti-rabbit or goat-anti-mouse horseradish peroxidase-conjugated IgG (1:1500, Abcam).
A Nuclear Protein Extraction Kit (Active Motif, Carlsbad, CA, USA) was used to extract the nuclear fractions according to the manufacturer’s instructions. The PVDF membranes were probed with specific antibodies: rabbit anti-β-catenin (1:1000, Abcam) and mouse anti-Lamin B1 (1:2000, Abcam) overnight at 4 °C. Then, they are followed by incubation with the second antibodies: goat-anti-rabbit or goat-anti-mouse horseradish peroxidase-conjugated IgG (1:1500, Abcam).
The target gene of miR-140-5p was detected by luciferase reporter gene system
Online software TargetScan (http://www.targetscan.org) was used to predict the target gene of miR-140-5p, and the results showed Wnt1 might be the target gene. The wild-type (WT) 3′-UTR of Wnt1 mRNA has a putative miR-140-5p-binding site. The mutant-type (MuT) 3′-UTR of Wnt1 was constructed. The WT 3′-UTR of Wnt1 and MuT 3′-UTR of Wnt1 were inserted into pMIRGLO vectors (Promega Corporation, Madison, WI, USA). The miR-140-5p mimic, miR-negative control, WT 3′-UTR of Wnt1 vector, and MuT 3′-UTR of Wnt1 vector were co-transfected into the HEK293 cells. Following cell transfection for 48 h, luciferase activity was detected using a dual-luciferase reporter assay system (E1910; Promega Corporation).
Statistical analysis
All the data were expressed by mean ± SD in this study and analyzed using statistical software SPSS 21.0. The difference among the groups was estimated by one-way ANOVA and compared using the LSD method. P < 0.05 was considered statistically significant.