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Journal of Peking University (Health Sciences) ›› 2023, Vol. 55 ›› Issue (4): 689-696. doi: 10.19723/j.issn.1671-167X.2023.04.020

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Characteristics and microRNA expression profile of exosomes derived from odontogenic dental pulp stem cells

Yu-yang YE,Lin YUE,Xiao-ying ZOU*(),Xiao-yan WANG*()   

  1. Department of Cariology and Endodontology, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices & Beijing Key Laboratory of Digital Stomatology & NHC Research Center of Engineering and Technology for Computerized Dentistry & NMPA Key Laboratory for Dental Materials, Beijing 100081, China
  • Received:2020-10-10 Online:2023-08-18 Published:2023-08-03
  • Contact: Xiao-ying ZOU,Xiao-yan WANG E-mail:zouxiaoying1125@163.com;wangxiaoyanpx@163.com

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Abstract:

Objective: To investigate the characteristics of exosomes derived from dental pulp stem cells (DPSCs) in the direction of odontogenic differentiation, to analyze the differences in microRNA expression profile between exosomes derived from undifferentiated and odontogenic DPSCs, and to analyze their possible signal transduction pathways. Methods: (1) DPSCs were cultured in α minimum Eagle' s medium (α-MEM), and odontogenic DPSCs were cultured in odontogenic differentiation medium for 21 days, using alizarin red staining and alkaline phosphatase staining to identify the odontogenic differentiation. Exosomes from the cell supernatant were isolated respectively, named as dental pulp stem cells-exosomes (DPSCs-Exo) and dental pulp stem cells-odontogenic-exosomes (DPSCs-OD-Exo). The exosomes were identified by transmission electron microscopy, nanoparticle tracking analysis and Western blot. (2) The microRNA expression profiles of DPSCs-Exo and DPSCs-OD-Exo were investigated by microRNA microarray. To validate the result of the microRNA microarray, real-time quantitative polymerase chain reaction (real-time PCR) assay was applied on 3 most significantly differential expressed microRNA. Pathway analysis was taken to detect enriched pathways associated with the predicted target genes of microRNA. Results: (1) The DPSCs were isolated and cultured in vitro showed typical fibroblast-like morphology. The odontogenic differentiated DPSCs were spindle-shaped, polygonal, and uniform in size. Odontogenic differentiation group showed a large number of dark deposits in alizarin red staining and the cells were darkly stained in alkaline phosphatase staining, while the cells in normal culture medium group did not show obvious dyeing. The DPSCs-Exo and DPSCs-OD-Exo had the same morphology, both showed bilayer membrane and cup-shape. The peak sizes of DPSCs-Exo and DPSCs-OD-Exo were (114.67±9.07) nm and (134.00±8.54) nm, respectively. The difference between the two was statistically significant. DPSCs-Exo and DPSCs-OD-Exo both expressed the markers of exosomes, tumor susceptibility gene (TSG)101 and CD63. (2) microRNA microarray results showed that the expression profiles of DPSCs-Exo and DPSCs-OD-Exo were different. Nineteen increased by more than two times, and one decreased by 64%. Real-time PCR results showed that the expression levels of microRNA-1246, microRNA-1246-100-5p and microRNA-1246-494-3p in DPSCs-OD-Exo were significantly up-regulated. The difference was statistically significant. microRNA target prediction database and gene signaling pathway database were used to analyze differentially expressed microRNA, and it was predicted that differentially expressed microRNA could target axis inhibition protein 2(AXIN2) gene and Wnt/β-catenin signaling pathway. Conclusion: DPSCs-OD-Exo and DPSCs-Exo had differences in their microRNA expression profile. Those differentially expressed microRNA may be involved in the regulation of DPSCs odontogenic differentiation.

Key words: Dental pulp stem cells, Exosomes, microRNA

CLC Number: 

  • R781.3

Table 1

microRNA reverse transcription primers"

GenesReverse transcription primers
microRNA-100-5p5′GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCACAAG3′
microRNA-12465′GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCCTGCT3′
microRNA-494-3p5′GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACGAGGTT3′
microRNA-191-5p5′GTCGTATCCAGTGCGTGTCGTGGAGTCGGCAATTGCACTGGATACGACCAGCTG3′

Table 2

microRNA bidirectional primers"

GenesBidirectional primersAnnealing temperature/℃Base pair/bp
microRNA-100-5pGSP: 5′GCAACCCGTAGATCCGAA3′
R: 5′CAGTGCGTGTCGTGGAGT3′		6062
microRNA-1246GSP: 5′GGGGAATGGATTTTTGG3′
R: 5′CAGTGCGTGTCGTGGAG3′		6063
microRNA-494-3pGSP: 5′GGGGTGAAACATACACGGGA3′
R: 5′GTGCGTGTCGTGGAGTCG3′		6064
microRNA-191-5pGSP: 5′GGCAACGGAATCCCAAAAG3′
R: 5′GTGCGTGTCGTGGAGTCG3′		6063

Figure 1

Morphology and differentiation identification results of undifferentiated and odontogenic DPSCs A, B, bright view of undifferentiated and odontogenic DPSCs cultured for 3 weeks; C, D, alizarin red staining results of undifferentiated and odontoge-nic DPSCs; E, F, alkaline phosphatase staining results of undifferentiated and odontogenic DPSCs. DPSCs, dental pulp stem cells."

Figure 2

Identification of DPSCs-Exo and DPSCs-OD-Exo A, B, DPSCs-Exo and DPSCs-OD-Exo exosomes morphology under transmission electron microscope; C, Western blot detects the expression of exosomal markers in DPSCs-Exo and DPSCs-OD-Exo; D, E, NTA results of DPSCs-Exo and DPSCs-OD-Exo. DPSCs-Exo, dental pulp stem cells-exosomes; DPSCs-OD-Exo, dental pulp stem cells-odontogenic-exosomes; TSG101, tumor susceptibility gene 101; NTA, nanoparti-cletracking analysis."

Table 3

Differential expressed microRNA in DPSCs-OD-Exo and DPSCs-Exo"

microRNAIncrease/decreaseFoldP
microRNA-100-5pIncrease2.16< 0.01
microRNA-1246Increase84.65< 0.01
microRNA-1587Increase2.03< 0.01
microRNA-3136-5pIncrease2.27< 0.01
microRNA-3912-5pIncrease2.69< 0.01
microRNA-3923Increase2.38< 0.01
microRNA-3945Increase2.31< 0.01
microRNA-4259Increase2.18< 0.01
microRNA-4707-5pIncrease2.15< 0.01
microRNA-4754Increase2.00< 0.01
microRNA-494-3pIncrease2.82< 0.01
microRNA-5196-5pIncrease2.18< 0.01
microRNA-5687Increase2.37< 0.01
microRNA-588Increase2.25< 0.01
microRNA-6133Increase2.35< 0.01
microRNA-6779-5pIncrease2.00< 0.01
microRNA-6813-5pIncrease2.10< 0.01
microRNA-6823-5pIncrease2.30< 0.01
microRNA-6832-5pIncrease2.21< 0.01
microRNA-1-3pDecrease2.19< 0.01

Figure 3

DPSCs-Exo and DPSCs-OD-Exo microRNA heat map microRNA levels in DPSCs-OD-Exo significantly changed when compared with that in DPSCs-Exo, the green to red corresponds to the gene expression level from low to high. DPSCs-Exo, dental pulp stem cells-exosomes; DPSCs-OD-Exo, dental pulp stem cells-odontogenic-exosomes."

Figure 4

microRNA-1246 was used as an example to predict target genes from the database miRTarBase, miRDB, miRWalk databases were used to predict micro-RNA-1246 target genes to make a Venn diagram."

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