The Suppressive Effects of Mir-508-5P on the Odontogenic
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Liu et al. Stem Cell Research & Therapy (2019) 10:35 https://doi.org/10.1186/s13287-019-1146-8 RESEARCH Open Access The suppressive effects of miR-508-5p on the odontogenic differentiation of human dental pulp stem cells by targeting glycoprotein non-metastatic melanomal protein B Fengxi Liu1,2†, Xin Wang3†, Yun Yang4, Rongrong Hu1,5, Wenhao Wang5 and Yuliang Wang1,5* Abstract Background: Although the involvement of glycoprotein non-metastatic melanomal protein B (GPNMB) in regulating the odontogenic differentiation of human dental pulp stem cells (hDPCs) has been identified, the underlying mechanisms are largely unknown. The purpose of this study is to investigate the effects of miR-508-5p on the GPNMB expression and the odontogenic differentiation of hDPCs. Methods: In this study, hDPCs were isolated and identified by flow cytometric analysis. Based on bioinformatics analysis, dual luciferase reporter assay was performed to verify GPNMB acting as a target of miR-508-5p. The regulatory roles of miR-508-5p in odontogenetic differentiation of hDPCs were investigated through its inhibition or overexpression (miRNA mimics and miRNA inhibitors). qRT-PCR and Western blot analysis were used to detect the expression of odontogenetic marker genes and proteins. The assays of alkaline phosphatase (ALP) activity and Alizarin Red S staining were performed to evaluate the odontogenetic phenotype. Results: We first found that the levels of miR-508-5p expression decreased gradually during odontogenesis of hDPCs, while the expressions of GPNMB were upregulated obviously. The suppressive effects of miR-508-5p on GPNMB were determined by oligonucleotide transfection in hDPCs and dual luciferase reporter assay in 293T cells. Subsequently, the significant inhibition of hDPC odontogenesis after the overexpression of miR-508-5p was observed, which is consistent with the decreased expression levels of several odontoblast-specific genes, such as dentin matrix protein 1 (DMP-1), dentin sialophosphoprotein (DSPP), and osteocalcin (OCN), as well as the decreased activity of ALP and weakened Alizarin Red S staining. Furthermore, ectopic expression of GPNMB (lacking 3′-UTR) rescued the effects of miR-508-5p on odontogenic differentiation. Conclusions: Our study demonstrated that miR-508-5p regulated the osteogenesis of hDPCs by targeting GPNMB and provided novel insight into the critical roles of microRNAs in hDPC differentiation. Keywords: Dental pulp stem cell, miRNA, GPNMB, Odontogenesis, Differentiation * Correspondence: [email protected] †Fengxi Liu and Xin Wang contributed equally to this work. 1Department of Oral and Maxillofacial Surgery, Yantai Affiliated Hospital of Binzhou Medical University, No 717, Jinbu Street, Muping District, Yantai 264100, People’s Republic of China 5College of Stomatology, Binzhou Medical University, Yantai 264003, People’s Republic of China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Liu et al. Stem Cell Research & Therapy (2019) 10:35 Page 2 of 10 Background miRNA 508-5p (miR-508-5p) as a potential regulator for Cell-based tissue engineering has become increasingly GPNMB. The level of miR-508-5p and GPNMB was de- important in regenerative medicine. Since the discovery tected during the odontogenic differentiation of hDPCs by Gronthos et al., human dental pulp stem cells and a negative correlation between miR-508-5p and (hDPCs) have been receiving more attention recently be- GPNMB was observed. The luciferase reporter assays cause of its some advantages, such as easy availability, further confirmed that GPNMB is a direct target of minimal invasion, multipotent ability, less immune rejec- miR-508-5p. Also, our results showed that the overex- tion, and avoidance of ethical concerns [1–3]. These out- pression and knockdown of miR-508-5p can influence standing features make the hDPCs as a suitable source the differentiation capacity of hDPCs remarkably of tissue repair, not only in dentinogenesis but also in followed by the expression changes of GPNMB. Further- regeneration-related diseases [4, 5]. Although tremen- more, the transfection of GPNMB without 3′-UTR re- dous advances about hDPCs have been achieved in the versed the effects of miR-508-5p on odontogenesis of past decade, the understandings of hDPCs are still insuf- hDPCs. These results provided evidence that ficient and its regulation mechanism is still not clear. miR-508-5p can inhibit the odontogenic differentiation MicroRNAs (miRNAs), a class of small non-coding of hDPCs by targeting GPNMB. RNAs that approximately 17 to 25 nucleotides in length, can regulate gene expression by recognizing the 3′ un- Materials and methods translated region (3′-UTR) of their target mRNAs lead- Isolation and culture of hDPCs ing to mRNA decay or translation repression [6]. It has All experimental procedures were approved by the Eth- been demonstrated that miRNAs play essential roles in a ics Committee of the School of Stomatology, Binzhou broad range of biological processes, including develop- Medical University. hDPCs were isolated and cultured ment, cell proliferation/apoptosis, signal pathways, in- according to our published work [15]. Briefly, extracted flammation, and tumors [7–9]. Similarly, miRNAs have healthy human third molars (n = 5) were collected from emerged as important regulators in stem cell biology, re- patients who were undergoing treatment at the Dental lated to cell reprogramming, maintenance of stemness, Department of the Yantai Affiliated Hospital of Binzhou and regulation of cell differentiation [10, 11]. Recently, Medical University. After gentle separation from the although the role of miRNAs in the proliferation and pulp chambers, the pulp tissues were minced into tiny differentiation of hDPCs has attracted more attention, pieces (about 1 mm) and then digested with a solution the limited understanding of the molecular mechanism of 3 mg/mL collagenase type I and 4 mg/mL dispase II underlying their differentiation is also a huge obstacle for 1 h at 37 °C. The cell suspension was seeded in a prior to the usage of hDPCs in cell therapy. 35-mm culture dish (Corning Costar, USA) and cultured Glycoprotein non-metastatic melanomal protein B in maintenance medium containing Dulbecco modified (GPNMB), also known as osteoactivin, is a type 1 trans- Eagle medium (DMEM) supplemented with 10% fetal membrane glycoprotein and expressed in a wide array of bovine serum (FBS) and 100 U/mL penicillin/strepto- tissues and plays regulatory roles in various cellular mycin (Sigma, St. Louis, MO, USA) at 37 °C in a hu- functions, including cell adhesion, migration, and differ- midified atmosphere of 5% CO2. The fresh medium was entiation [12]. Thus, GPNMB has been implicated in changed every 2 days. Approximately 10 days after seed- physiological and pathophysiological cascades of tissue ing, the cells became nearly confluent with the cell num- injury and repair. Since the first report that GPNMB ber about 2–3×105 (passage 0). The cell population was played an essential role in osteoblast differentiation and detached and transferred to 6-well plates (passage 1) matrix mineralization, increasing evidence suggested (Corning Costar, USA). Cells were passaged at the ratio that GPNMB can served as an osteoblast-specific gene of 1:3 when they reached 75–85% confluence. Cells from in the bone and play a vital role for osteoblast differenti- the third passage (P3) were used for all experiments. ation [13, 14]. As the great similarity between osteogenic The multipotent differentiation potential of the hDPCs and odontogenic differentiation, our findings and other was identified by osteogenic and adipogenic differenti- published studies have also demonstrated the involve- ation induction. Briefly, the cells were exposed to osteo- ment of GPNMB in regulating the odontogenic differen- genic medium (DMEM supplemented with 10 nmol/L tiation of hDPCs [15]. However, the mechanisms dexamethasone, 10 mmol/L β-glycerophosphate, 50 μg/ involving the regulation of GPNMB during the odonto- mL ascorbate phosphate, 10 nmol/L 1,25-dihydroxyvita- genic differentiation of hDPCs are still unclear. min D3, and 10% FBS) and adipogenic medium (DMEM In this study, we tested the hypothesis that the supplemented with 1 mM dexamethasone, 10 mg/mL changes of GPNMB expression can be regulated in the insulin, 100 mM indomethacin, 0.5 mM methylisobutyl- miRNA level during the odontogenesis of hDPCs. Bio- zanthine, and 10% FBS) for 14 days. Alizarin Red S and informatics analysis was first performed to reveal Oil Red O reagent were used to visualize the calcium Liu et al. Stem Cell Research & Therapy (2019) 10:35 Page 3 of 10 accumulation and oil droplets, respectively. After stain- (Invitrogen, USA). Cells were harvested after 48 h for ing, the cells were imaged by an inverted light micro- the analysis of GPNMB expression. scope (Olympus, Japan). To estimate whether miR-508-5p could bind to the For odontoblastic induction, hDPCs were plated in GPNMB 3′-UTR, 293T cells were seeded in 96-well 6-well plates (Corning Costar, USA) at an initial density plates (Corning Costar, USA). The cells were transfected of 2 × 105 cells/well and cultured in DMEM supple- with the GPNMB-Wt or GPNMB-Mt reporter plasmid, mented with 10% FBS, antibiotics, 50 mg/mL ascorbic miR-508-5p-negative controls (NC, 50 nM), negative acid, 10 mmol/L sodium β-glycerophosphate, and controls for miR-508-5p siRNA (NC-Si, 50 nM), 10 nmol/L dexamethasone (Sigma, St. Louis, MO, USA) miR-508-5p mimics (50 nM), or miR-508-5p inhibitors for 14 days.