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DMP1 Is a Target of Let-7 in Dental Pulp Cells

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DMP1 Is a Target of Let-7 in Dental Pulp Cells INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 30: 295-301, 2012 DMP1 is a target of let-7 in dental pulp cells JING YUE, BULING WU, JIE GAO, XIN HUANG, CHANGXIA LI, DANDAN MA and FUCHUN FANG Department of Stomatology, Nanfang Hospital, College of Stomatology, Southern Medical University, Guangzhou, P.R. China Received February 2, 2012; Accepted April 5, 2012 DOI: 10.3892/ijmm.2012.982 Abstract. Members of the let-7 family have been shown to play nant disorder of dentin formation, has been also mapped to a critical role in cell differentiation and tumorigenesis. However, the same region of the genome, indicating that DMP1 expres- potential targets of let-7 are still unclear. In the current study, sion is tightly linked genetically to its disease phenotype (9). we used bioinformatic analysis combined with DNA sequence Accordingly, DMP1 has been shown to play a prime role in analysis to identify potential let-7 targets. We discovered that dentin mineralization (10,11). Dentin and bone extracellular dentin matrix protein 1 (DMP1), which is a non-collagenous matrix (ECM) were shown to contain both the full length protein essential in the mineralization of dentin and bone, has DMP1 as well as its processed N-terminal (N-ter) (37 kDa) a let-7 binding site in its 3'-untranslated region. Furthermore, and C-terminal (C-ter) (57 kDa) fragments. It was shown to reporter assays demonstrated that the DMP1 3'-untranslated regulate the transcription of DSPP during early odontoblast region can be regulated directly by the members of let-7. Gene differentiation through binding of the promoter region through expression levels of let-7 and DMP1 were validated by qRT-PCR its carboxyl end (residues 420-489) and was implicated in of dental pulp cells cultured in a mineralizing medium. Our signaling functions (12). This result led us to the hypothesis results suggest that DMP1 is regulated post-transcriptionally that DMP1 may have functions other than regulating miner- by let-7 during odontoblast differentiation. alization. Recently, it was suggested that DMP1 belongs to the SIBLING (small integrin binding ligand N-linked Introduction glycoprotein) family of extracellular matrix proteins (13). The expression of SIBLING family proteins such as osteopontin Dental pulp cells (DPCs) have the potential to be mineral- and bone sialoprotein in soft tissues has been reported. DMP1 ized, which plays a key role in pulp repair and dentinogenesis. gene expression was also detected in fetal bovine brain by Reparative dentin formation and pulp regeneration after partial northern analysis and in newborn mouse brain by in situ degradation are under the control of pulp progenitor cells (1). hybridization (14,15). These reports raised the possibility of Dentinogenesis is regulated by tissue interactions associated DMP1 expression in other soft tissues, such as liver, muscle, with complicated signal pathways. Dentin matrix protein 1 kidney, pancreas, salivary and eccrine sweat glands. Together (DMP1), an essential noncollagenous and acidic phosphory- with results obtained in previous reports, it is suggested that, lated extracellular matrix protein, is highly expressed in tooth in addition to its high affinity to calcium or hydroxyapatite odontoblasts and bone osteocytes, with low level expression in due to its acidic character and to its role in the mineralization osteoblasts and cartilage (2-4). It is a multifunctional protein process, DMP1 may affect various cell activities. involved in the biomineralization of bones and dentin (5), Cells contain a variety of noncoding RNAs, such as phosphate homeostasis, and differentiation of odontoblasts microRNAs (miRNAs), which are small (22-nt) endogenous and osteoblasts (6). The DMP1 gene has been mapped to noncoding RNAs that anneal to the 3' untranslated region human chromosome 4q21:22 (7) (chromosome 5q21 in mice) (3'UTR) of target mRNAs to inhibit translation and lower (8). Dentinogenesis imperfecta type II, the autosomal domi- protein levels. It remains to be established how specific miRNAs contribute to regulate the onset of a tissue-specific phenotype in response to a multifunctional morphogen. Let-7 was one of the first identified miRNA families, consisting of 12 closely related genes in which each isomer is usually located Correspondence to: Professor Buling Wu, Department of on a different chromosome and which is highly conserved Stomatology, Nanfang Hospital, College of Stomatology, Southern across animal species in sequence and timing of expression Medical University, 1838 N. Guangzhou Ave., Guangzhou 510515, P. R. Ch i na (16,17). Let-7 plays a significant role in cell proliferation, E-mail: [email protected] differentiation and oncogenesis; identification of the target genes of miRNA may help to characterize its diverse functions Key words: dentin matrix protein 1, dual luciferase reporter assay, (17,18). By bioinformatic analysis, we have found a potential dental pulp cells, let-7 binding site for let-7 within the 3'UTR of DMP1. It is important to understand the molecular mechanism and specific genes during the developmental process. It has been proven that DMP1 is regulated by many growth 296 YUE et al: DENTIN MATRIX PROTEIN 1 and transcription factors in previous studies. However, the their targeted prediction by all three programs, conser vation regulation of DMP1 is not fully understood, particularly the of the binding region, and strength of the predicted interaction. understanding of its post-transcriptional regulation. Here we present the probable miRNA pathway of DMP1 in its post- Dual luciferase reporter gene construct. A 1041-bp fragment transcriptional regulation. Sophisticated computer-based of the DMP1 3'UTR containing the predicted binding site for prediction approaches of microRNAs and of their targets, let-7 was amplified from human genomic DNA using primers and effective biological validation techniques for validating with a short extension containing cleavage sites for XhoI these predictions, now play a central role in discovery of (5' end) and NotI (3' end): DMP1 forward (5'-CCGCTCGAGC microRNAs and elucidating their functions. In this study, we ATCAGCTGTCCTAAGAAGCAGTT-3') and DMP1 reverse identified that let-7 may be the potential regulatory miRNA of (5'-ATAAGAATGCGGCCGCTTTCTTCTGGGTATTATAA DMP1 gene through utilization of bioinformatics analysis and TCTTTATTAC-3'). Amplicons were cleaved with XhoI and a dual luciferase reporter assay. In addition, we assessed the NotI and cloned in between the XhoI and NotI cleavage sites of expression levels of let-7 at 10 and 21 days of differentiation in the psi-CHECK2 luciferase vector (Promega) downstream of odonto- and osteoblasts in dental pulp cells. the Renilla luciferase reporter gene. Materials and methods Dual luciferase reporter assay. Luciferase assays were performed using the Dual-Luciferase assay kit as previously Subjects and cell culture. Normal human impacted third described (Promega). For let-7 target validation, 293-T cells molars were collected from adults (16-24 years of age) at were grown to a cell density of 60-70% and co-transfected the Nanfang Hospital in the Southern Medical University. in 24-well plates with the indicated psiCHECK2 lucif- The study protocol was approved by the Institutional Ethics erase construct (0.5 µg/well) and miRNA (20 µM) using Committee, and informed consent was obtained from all Lipofectamine 2000. Following 48 h, the cells were harvested patients. DPCs were isolated and subjected to odontogenic in passive lysis buffer, and luciferase activity was measured induction as previously reported (19). Tooth surfaces were with a GloMax™ 20/20 luminometer (Promega). Renilla cleaned and cut around the cementum-enamel junction by luciferase activity was normalized to corresponding firefly using sterilized dental fissure burs to reveal the pulp chamber. luciferase activity and plotted as a percentage of the control The pulp tissue was gently separated from the crown and root between samples. Luciferase experiments were measured in and then digested in a solution of 3 mg/ml collagenase type I triplicate using independent samples, as indicated. and 4 mg/ml dispase for 30-60 min at 37˚C. The cells were cultured in a growth medium containing Dulbecco's modi- qRT-PCR. Dental pulp cells cultured in a mineralizing medium fied Eagle's medium (DMEM) (Gibco) with 15% fetal bovine were able to differentiate into odontoblast-like cells. Samples serum, 100 U/ml penicillin and 100,000 µg/ml streptomycin, were harvested for the isolation of RNA at 10 and 21 days of and then cultured at 37˚C in 5% CO2. DPCs at passage 3 differentiation to detect quantitative changes in gene DMP1 were cultured in DMEM with 15% FBS until they reached and three miRNAs. Cells were cultured in a growth medium 70-80% confluence. Cells were then induced in odontogenic that served as the control. Total-RNA was extracted from cells medium consisting of 50 mg/ml ascorbic acid, 10 mM using the TRIzol reagent (Invitrogen, Carlsbad, CA) per the β-glycerophosphate, and 0.01 mM dexamethasone (Sigma) in manufacturer's instructions. RNA was analyzed for integrity, DMEM with 15% FBS for 7-21 days. Control samples were purity and concentration by gel electrophoresis and spectro- DPCs grown in DMEM with 15% FBS and harvested at 80% photometry and stored at -80˚C until analysis. confluence. T293 cells were grown in DMEM (Gibco) supple- For quantitative RT-PCR analysis, 0.1 µg of RNA per mented with 10% fetal bovine serum. reaction was used with the QuantiTech SYBR-Green RT-PCR kit and primers specific for DMP1. To quantify miRNA Mineralization staining. Mineralization of cultured DPCs was expression, total-RNA was reverse transcribed for use in two- determined using Alizarin Red (AR) staining. After Day 21, step quantitative RT-PCR using the stem-loop method. The the cell layer was washed with PBS and fixed in 10% formal- resulting cDNA was subjected to real-time qRT-PCR using dehyde (Sigma-Aldrich) at room temperature for 15 min, then the universal reverse primer in conjunction with a sequence- washed in duplicate with excess dH2O prior to the addition of specific forward primer for hsa-let-7a, hsa-let-7c, hsa-let-7d, 1 ml of 40 mM AR (pH 4.1).
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