Normal human impacted third molars were collected from adults (16–24 years of age) at the Nanfang Hospital in the Southern Medical University. The study protocol was approved by the Institutional Ethics Committee, and informed consent was obtained from all patients. DPCs were isolated and subjected to odontogenic induction as previously reported (19). Tooth surfaces were cleaned and cut around the cementum-enamel junction by using sterilized dental fissure burs to reveal the pulp chamber. The pulp tissue was gently separated from the crown and root and then digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase for 30–60 min at 37°C. The cells were cultured in a growth medium containing Dulbecco’s modified Eagle’s medium (DMEM) (Gibco) with 15% fetal bovine serum, 100 U/ml penicillin and 100,000 μg/ml streptomycin, and then cultured at 37°C in 5% CO₂. DPCs at passage 3 were cultured in DMEM with 15% FBS until they reached 70–80% confluence. Cells were then induced in odontogenic medium consisting of 50 mg/ml ascorbic acid, 10 mM β-glycerophosphate, and 0.01 mM dexamethasone (Sigma) in DMEM with 15% FBS for 7–21 days. Control samples were DPCs grown in DMEM with 15% FBS and harvested at 80% confluence. T293 cells were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum.

Mineralization of cultured DPCs was determined using Alizarin Red (AR) staining. After Day 21, the cell layer was washed with PBS and fixed in 10% formaldehyde (Sigma-Aldrich) at room temperature for 15 min, then washed in duplicate with excess dH₂O prior to the addition of 1 ml of 40 mM AR (pH 4.1). The plates were incubated at room temperature for 30 min under gentle shaking. Following aspiration of the unincorporated dye, the plates were washed twice with dH₂O and visualized using phase microscopy (Nikon).

DMP1 was identified as a potential target to search for miRNAs. Three different miRNA target prediction programs were used, TargetScan (http://www.targetscan.org/), the UCSC genome browser tract for PicTar4 (http://genome.ucsc.edu/), and the miRBase for miRanda (http://microrna.sanger.ac.uk/sequences/) (20–22). Each of these programs were searched for complementarity to the miRNA seed region in the 3′UTRs of DMP1. miRNAs were chosen based upon their targeted prediction by all three programs, conser vation of the binding region, and strength of the predicted interaction.

A 1041-bp fragment of the DMP1 3′UTR containing the predicted binding site for let-7 was amplified from human genomic DNA using primers with a short extension containing cleavage sites for XhoI (5′ end) and NotI (3′ end): DMP1 forward (5′-CCGCTCGAGC ATCAGCTGTCCTAAGAAGCAGTT-3′) and DMP1 reverse (5′-ATAAGAATGCGGCCGCTTTCTTCTGGGTATTATAA TCTTTATTAC-3′). Amplicons were cleaved with XhoI and NotI and cloned in between the XhoI and NotI cleavage sites of the psi-CHECK2 luciferase vector (Promega) downstream of the Renilla luciferase reporter gene.

Luciferase assays were performed using the Dual-Luciferase assay kit as previously described (Promega). For let-7 target validation, 293-T cells were grown to a cell density of 60–70% and co-transfected in 24-well plates with the indicated psiCHECK2 luciferase construct (0.5 μg/well) and miRNA (20 μM) using Lipofectamine 2000. Following 48 h, the cells were harvested in passive lysis buffer, and luciferase activity was measured with a GloMax™ 20/20 luminometer (Promega). Renilla luciferase activity was normalized to corresponding firefly luciferase activity and plotted as a percentage of the control between samples. Luciferase experiments were measured in triplicate using independent samples, as indicated.

Dental pulp cells cultured in a mineralizing medium were able to differentiate into odontoblast-like cells. Samples were harvested for the isolation of RNA at 10 and 21 days of differentiation to detect quantitative changes in gene DMP1 and three miRNAs. Cells were cultured in a growth medium that served as the control. Total-RNA was extracted from cells using the TRIzol reagent (Invitrogen, Carlsbad, CA) per the manufacturer’s instructions. RNA was analyzed for integrity, purity and concentration by gel electrophoresis and spectrophotometry and stored at −80°C until analysis.

For quantitative RT-PCR analysis, 0.1 μg of RNA per reaction was used with the QuantiTech SYBR-Green RT-PCR kit and primers specific for DMP1. To quantify miRNA expression, total-RNA was reverse transcribed for use in two-step quantitative RT-PCR using the stem-loop method. The resulting cDNA was subjected to real-time qRT-PCR using the universal reverse primer in conjunction with a sequence-specific forward primer for hsa-let-7a, hsa-let-7c, hsa-let-7d, hsa-let-7f, hsa-let-7g and hsa-let-7i. Each sample was performed in triplicate, and the results were normalized using primers to 18S rRNA (for DMP1) or U6 (for miRNA analysis) (Table I). The reactions were incubated at 95°C for 5 min for one cycle and then 95°C/15 sec, 65°C/15 sec, 72°C/32 sec for 40 cycles, with a final 10-min extension at 65°C. Results are expressed as fold change in expression relative to the control sample calculated using the equation RQ=2^−ΔΔCt.

Statistical analysis was performed with SPSS for Windows (version 13.0) using the ANOVA test. All data are presented as the mean ± SD (n≥3). P≤0.01 was considered statistically significant.

It has been demonstrated that dental pulp cells can be expanded as a dentin-pulp-like structure. Our results show that DPCs have the potential to differentiate into odontoblast-like cells when cultured in the culture medium contain dexamethasone (Dex) and/or β-glycerophosphate (β-GP) (23). Ten days later, mineralized nodules formed and became more condensed. Alizarin Red staining of mineralized nodules in representative cell cultures is demonstrated at 21 days in Fig. 1B.

With DMP1 being identified as a preferential target, miRNA target prediction was carried out using a combination of the following computational algorithms: TargetScan, miRanda, and PicTar. Based on the stem-loop feature of the miRNA and cross-species comparison, a number of computational algorithms have been developed to predict miRNAs from the genome. Among miRNAs being predicted to target DMP1, let-7 was complementary to multiple sequences of potential miRNAs with a high probability of binding to the 3′UTR of DMP1. To increase our probability of identifying miRNAs capable of regulating DMP1 post-transcriptionally, we selected to further examine eight miRNAs that were identified by all three search algorithms.

We used a luciferase reporter assay to test whether the 3′UTR of DMP1 contained sequences capable of interacting with let-7. The potential binding sites of let-7 within DMP1 3′UTR were obtained by TargetScan and PicTar. Synthetic oligos including predicted binding sites were annealed then cloned into XhoI/NotI site of psiCHECK-2. 293-T cells were transiently transfected with the relevant DMP1 3′UTR fragment and microRNA using Lipofectamine 2000 (Invitrogen) at the indicated concentrations.

To test whether the luciferase assay responds to let-7, we then tested reporters in which the 3′UTR of DMP1 was inserted downstream of firefly luciferase. The 3′UTR of DMP1 contains conserved let-7 seed matches at positions 988–994. When adding a miRNA that is able to interact with DMP1 3′UTR, the bioluminescence reaction of Photinus pyralis luciferase was annihilated, as miRNA inhibits translation of vector mRNA. This construct allowed us to quickly and quantitatively evaluate the miRNA’s effect on the 3′UTR of DMP1. The DMP1 3′UTR fragment was inserted into the psiCHECK2 luciferase vector. The relative luciferase activity in 293-T cells transfected with the luciferase vector alone was set at 100% for comparison. The binding ability of the eight predicted let-7 miRNAs with DMP1 is shown in Fig. 2.

Let-7a, let-7c, let-7d, let-7f, let-7g and let-7i (Fig. 3) all significantly (P<0.01) reduced luciferase activity when compared to the negative scrambled miRNA and the luciferase vector alone, while let-7b and let-7e (Fig. 4) did not. There are 6 members of let-7 family miRNAs predicted by the biology software TargetScan and miRanda, and 5 by PicTar4. Let-7 family is considered a potentially important regulator of DMP1 3′UTR.

To validate the above data, the expression of 6 miRNA genes in the process of dental pulp cell differentiation was determined using qRT-PCR analysis. The DMP1 transcript was predominantly expressed in odontoblasts and transiently in preameloblasts along with an involvement with odontoblast differentiation and mineralization, which is used as an indicator of odontoblastic differentiation. Dental pulp cells cultured in mineral izing medium exhibited odontoblastic features, including increasing DMP1. In our study, expression of DMP1 was weak before Day 0, but the amount increased by Day 10. After Day 21, DMP1 was strongly expressed. As a regulator of DMP1, the members of let-7 family need to exist in undifferentiated and differentiated dental pulp cells. Expression of 3 miRNAs on Day 10 was decreased, especially that of let-7a and let-7c. On Day 21, expression level of the 3 miRNAs was significantly decreased when DMP1 was at its peak. The expression of the other 3 miRNAs was higher in differentiated than undifferentiated cells, but the relative expression of let-7i rose higher than that of the other two miRNAs (Fig. 5).