Abstract
Background:
This study aims to clarify the mechanism underlying dental pulp cells-extracellular vesicles (DPC–EVs) carrying runt-related transcription factor 3 (RUNX3) in mediating odontogenic differentiation of dental pulp stem cells (DPSCs) with the involvement of miR-30a-5p-regulated NOTCH1.
Methods:
Extracellular vesicles (EVs) were isolated from human DPSCs, and identified using transmission electron microscopy, and nanoparticle tracking analysis. PBS, EVs, or EV inhibitor GW4869 was added to DPSCs for co-culture, whilst odontogenic differentiation was assessed in terms of ratio of mineralized nodules and expression odontoblast differentiation markers. Dual luciferase reporter gene assay and chromatin immunoprecipitation for binding relation among RUNX3, miR-30a-5p and NOTCH1were employed to evaluate their roles in odontogenic differentiation was determined. Animal experiment was established to confirm the effect of DPC-EVs-loaded RUNX3 on dental pulp.
Results:
In vitro finding demonstrated that EVs delivered RUNX3 to DPSCs, thereby activated miR-30a-5p expression and inhibited NOTCH1 expression, which was reversed by addition of GW4869. RUNX3 upregulation promoted miR-30a-5p while miR-30a-5p targeted and inhibited NOTCH1. Silencing of RUNX3 in EVs decreased expression of those differentiation markers, downregulated miR-30a-5p and upregulated NOTCH1.
Conclusion:
DPSC-EVs can carry RUNX3 to the DPSCs, promote the transcription of miR-30a-5p, and then inhibit the expression of NOTCH1, and finally promote the odontogenic differentiation of DPSCs.
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Data availability statement
The datasets generated and/or analysed during the current study are available in the manuscript and supplementary materials.
References
Huang GT-J, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009;88:792–806.
Yu D, Zhao X, Cheng JZ, Wang D, Zhang HH, Han GH. Downregulated microRNA-488 enhances odontoblast differentiation of human dental pulp stem cells via activation of the p38 MAPK signaling pathway. J Cell Physiol. 2019;234:1442–51.
Yu J, He H, Tang C, Zhang G, Li Y, Wang R, et al. Differentiation potential of STRO-1+ dental pulp stem cells changes during cell passaging. BMC Cell Biol. 2010;11:32.
Han N, Zheng Y, Li R, Li X, Zhou Mi, Niu Y, et al. β-catenin enhances odontoblastic differentiation of dental pulp cells through activation of Runx2. PLoS One. 2014;9:e88890.
Tatischeff I. Dictyostelium: A model for studying the extracellular vesicle messengers involved in human health and disease. Cells. 2019;8:225.
Sun G, Li G, Li D, Huang W, Zhang R, Zhang H, et al. hucMSC derived exosomes promote functional recovery in spinal cord injury mice via attenuating inflammation. Mater Sci Eng C Mater Biol Appl. 2018;89:194–204.
Alcayaga-Miranda F, Varas-Godoy M, Khoury M. Harnessing the angiogenic potential of stem cell-derived exosomes for vascular regeneration. Stem Cells Int. 2016;2016:3409169.
Narayanan K, Kumar S, Padmanabhan P, Gulyas B, Wan ACA, Rajendran VM, et al. Lineage-specific exosomes could override extracellular matrix mediated human mesenchymal stem cell differentiation. Biomaterials. 2018;182:312–22.
Huang CC, Narayanan R, Alapati S, Ravindran S. Exosomes as biomimetic tools for stem cell differentiation: applications in dental pulp tissue regeneration. Biomaterials. 2016;111:103–15.
Zhou H, Li X, Yin Y, He XT, An Y, Tian BM, et al. The proangiogenic effects of extracellular vesicles secreted by dental pulp stem cells derived from periodontally compromised teeth. Stem Cell Res Ther. 2020;11:110.
Barile L, Vassalli G. Exosomes: therapy delivery tools and biomarkers of diseases. Pharmacol Ther. 2017;174:63–78.
Wang Y, Feng Q, Ji C, Liu X, Li L, Luo J. RUNX3 plays an important role in mediating the BMP9-induced osteogenic differentiation of mesenchymal stem cells. Int J Mol Med. 2017;40:1991–9.
Bushweller JH. Targeting transcription factors in cancer—from undruggable to reality. Nat Rev Cancer. 2019;19:611–24.
Zheng L, Iohara K, Ishikawa M, Into T, Takano-Yamamoto T, Matsushita K, et al. RUNX3 negatively regulates Osterix expression in dental pulp cells. Biochem J. 2007;405:69–75.
Liu Z, Chen L, Zhang X, Xia X, Xing H, Zhang Y. RUNX3 rgulates vimentin expression via miR-30a during epithelial-mesenchymal transition in gastric cancer cells. J Cell Mol Med. 2014;18:610–23.
Liu L, Wang T, Huang D, Song D. Comprehensive analysis of differentially expressed genes in clinically diagnosed irreversible pulpitis by multiplatform data integration using a robust rank aggregation approach. J Endod. 2021;47:1365–75.
Che M, Gong W, Zhao Y, Liu M. Long noncoding RNA HCG18 inhibits the differentiation of human bone marrow-derived mesenchymal stem cells in osteoporosis by targeting miR-30a-5p/NOTCH1 axis. Mol Med. 2020;26:106.
Chen Z, Zhang K, Qiu W, Luo Y, Pan Y, Li J. Genome-wide identification of long noncoding RNAs and their competing endogenous RNA networks involved in the odontogenic differentiation of human dental pulp stem cells. Stem Cell Res Ther. 2020;11:114.
Fujii S, Fujimoto K, Goto N, Kanawa M, Kawamoto T, Pan H, et al. Characteristic expression of MSX1, MSX2, TBX2 and ENTPD1 in dental pulp cells. Biomed Rep. 2015;3:566–72.
Sun DG, Xin BC, Di W, Zhou L, HongBin W, Gong W. miR-140-5p-mediated regulation of the proliferation and differentiation of human dental pulp stem cells occurs through the lipopolysaccharide/toll-like receptor 4 signaling pathway. Eur J Oral Sci. 2017;125:419–25.
Zhang C, Chang J, Sonoyama W, Shi S, Wang CY. Inhibition of human dental pulp stem cell differentiation by Notch signaling. J Dent Res. 2008;87:250–5.
Zhan FL, Liu XY, Wang XB. The role of MicroRNA-143-5p in the differentiation of dental pulp stem cells into odontoblasts by targeting Runx2 via the OPG/RANKL signaling pathway. J Cell Biochem. 2018;119:536–46.
Mozaffari MS, Emami G, Khodadadi H, Baban B. Stem cells and tooth regeneration: prospects for personalized dentistry. EPMA J. 2019;10:31–42.
Casagrande L, Cordeiro MM, Nör SA, Nör JE. Dental pulp stem cells in regenerative dentistry. Odontology. 2011;99:1–7.
Xuan K, Li B, Guo H, Sun W, Kou X, He X, et al. Deciduous autologous tooth stem cells regenerate dental pulp after implantation into injured teeth. Sci Transl Med. 2018;10:eaaf3227.
Huang X, Xiong X, Liu J, Zhao Z, Cen X. MicroRNAs-containing extracellular vesicles in bone remodeling: an emerging frontier. Life Sci. 2020;254:117809.
Xiaoli H, Zhong Y, Kong Y, Chen Y, Feng J, Zheng J. Lineage-specific exosomes promote the odontogenic differentiation of human dental pulp stem cells (DPSCs) through TGFβ1/smads signaling pathway via transfer of microRNAs. Stem Cell Res Ther. 2019;10:170.
Hecht J, Stricker S, Wiecha U, Stiege A, Panopoulou G, Podsiadlowski L, et al. Evolution of a core gene network for skeletogenesis in chordates. PLoS Genet. 2008;4:e1000025.
Zhang J, Nanwei Xu, Changlin Yu, Miao K, Wang Q. LncRNA PART1/miR-185-5p/RUNX3 feedback loop modulates osteogenic differentiation of bone marrow mesenchymal stem cells. Autoimmunity. 2021;54:422–9.
Malizia AP, Wang DZ. MicroRNAs in cardiomyocyte development. Wiley Interdiscip Rev Syst Biol Med. 2011;3:183–90.
Song J, Kim D, Jin EJ. MicroRNA-488 suppresses cell migration through modulation of the focal adhesion activity during chondrogenic differentiation of chick limb mesenchymal cells. Cell Biol Int. 2011;35:179–85.
Peng W, Zhu S, Chen J, Wang J, Rong Q, Chen S. Hsa_circRNA_33287 promotes the osteogenic differentiation of maxillary sinus membrane stem cells via miR-214-3p/Runx3. Biomed Pharmacother. 2019;109:1709–17.
Zeng L, Zhao N, Li F, Han D, Liu Y, Liu H, et al. miR-675 promotes odontogenic differentiation of human dental pulp cells by epigenetic regulation of DLX3. Exp Cell Res. 2018;367:104–11.
Wang BL, Wang Z, Nan X, Zhang QC, Liu W. Downregulation of microRNA-143-5p is required for the promotion of odontoblasts differentiation of human dental pulp stem cells through the activation of the mitogen-activated protein kinases 14-dependent p38 mitogen-activated protein kinases signaling pathway. J Cell Physiol. 2019;234:4840–50.
Bhome R, Del Vecchio F, Lee GH, Bullock MD, Primrose JN, Sayan AE, et al. Exosomal microRNAs (exomiRs): small molecules with a big role in cancer. Cancer Lett. 2018;420:228–35.
Goto N, Fujimoto K, Fujii S, Ida-Yonemochi H, Ohshima H, Kawamoto T, et al. Role of MSX1 in osteogenic differentiation of human dental pulp stem cells. Stem Cells Int. 2016;2016:8035759.
Yang C, Jia R, Zuo Q, Zheng Y, Wu Q, Luo B, et al. microRNA-143–3p regulates odontogenic differentiation of human dental pulp stem cells through regulation of the osteoprotegerin-RANK ligand pathway by targeting RANK. Exp Physiol. 2020;105:876–85.
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We would like to give our sincere appreciation to the reviewers for their helpful comments on this article.
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This study was approved by the was approved by the Ethics Committee of The People's Hospital of Suzhou New District. All procedures performed in studies were in accordance with the ethical standards of the Ethics Committee of The People's Hospital of Suzhou New District (approval number: 2022-027) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All patients have ambush teeth, and provided informed consent to use their teeth for scientific research. All nude mice experiments are in accordance with the agreement approved by the UIC Animal Care Committee. This study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
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Chi, Y., Liu, T., Jin, Q. et al. Extracellular Vesicles Carrying RUNX3 Promote Differentiation of Dental Pulp Stem Cells. Tissue Eng Regen Med 21, 111–122 (2024). https://doi.org/10.1007/s13770-023-00578-1
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DOI: https://doi.org/10.1007/s13770-023-00578-1