Int J Stomatol

Int J Stomatol ›› 2022, Vol. 49 ›› Issue (1): 27-36.doi: 10.7518/gjkq.2022026

• Orginal Article • Previous Articles     Next Articles

MicroRNA profile of exosomes derived from dental pulp stromal cells under three-dimensional culture condition

Ai Xiaoqing(),Dou Lei,Qiao Xin,Yang Deqin()   

  1. Dept. of Cariology and Endodontics, Stomatological Hospital of Chong-qing Medical University, Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 401147, China
  • Received:2021-04-05 Revised:2021-10-14 Online:2022-01-01 Published:2022-01-07
  • Contact: Deqin Yang E-mail:2018110850@stu.cqmu.edu.cn;yangdeqin@hospital.cqmu.edu.cn
  • Supported by:
    This study was supported by National Natural Science Foundation of China(31970783);National Natural Science Foundation of China Youth Fund(81800958)

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Abstract: Objective This study aims to analyze and compare microRNA (miRNA) profiles in exosomes (Exo) derived from human dental pulp stromal cells (DPSCs) under two-dimensional (2D) and three-dimensional (3D) culture condition. Methods DPSC-Exo was extracted from 2D and 3D culture of DPSCs and identified by transmission electron microscopy, Western blot analysis, and nanoparticle tracking analysis (NTA). Differentially expressed miRNAs were screened by high-throughput sequencing for bioinformatics analysis and target gene prediction related to tissue regeneration and repair by using Dr.Tom system and TargetScan Website. Results 2D-DPSC-Exo and 3D-DPSC-Exo showed “saucer-like” double-layer membrane structure, expressed CD63 and CD9 positively and had diameter consistent with Exo characteristics. A total of 253 Exo-derived miRNAs were detected, among which 222 were in the 3D group and 99 were specifically expressed. Compared with the 2D culture, a significant difference of 60 [︱log2(3D/2D)︱≥1, Qvalue≤0.001]. Differentially expressed miRNAs were mainly involved in cellular process and binding. The Kyoto encyclopedia of genes and genomes (KEGG) pathway showed that these miRNAs were significantly enriched in metabolic pathways. Candidate target genes of miR-302 include fibroblast growth factor (FGF) 19 and epidermal growth factor receptor. Candidate target genes of miR-24-3p may play a role in tissue regeneration and repair, including neuronal differentiation (NEUROD) 2, neuroepithelial cell transforming 1, NEUROD1, neuronal regeneration-related protein, FGF11, FGF binding protein 3, FGF receptor 3, platelet-derived growth factor receptor (PDGFR) beta polypeptide, PDGFR alpha polypeptide, angiopoietin 4, and insulin-like growth factor binding protein 5. Conclusion Compared with 2D culture, 3D culture could regulate the expression of some miRNAs in DPSC-Exo. Upregulated miRNAs are mainly related to regeneration and repair, and 3D culture might be suitable to optimize the therapeutic potential of Exo.

Key words: three-dimensional culture, dental pulp stromal cells, exosomes sequencing, microRNA


TrendMD: 

Fig 1

Culture and identifite of DPSCs and Exo under 2D and 3D culture conditions"

Fig 2

Differential miRNA expression"

Tab 1

Partial up-regulated miRNA"

基因ID 2D组表达量 3D组表达量 log2(3D/2D) Qvalue
hsa-miR-302a-3p 26 978 5.23 1.79e-221
hsa-miR-302b-3p 192 1 578 3.04 2.85e-111
hsa-miR-302c-3p 16 309 4.27 1.04e-52
hsa-miR-302d-3p 38 1 017 4.74 2.80e-201
hsa-miR-24-3p 17 702 5.37 1.23e-92
hsa-miR-27b-3p 363 989 1.44 8.07e-28
hsa-miR-34a-5p 942 2 453 1.38 1.28e-25
hsa-miR-100-5p 557 1 310 1.23 8.61e-24

Fig 3

Significantly differentially expressed miRNAs in 2D DPSC-Exo and 3D DPSC-Exo"

Tab 2

TargetScan website predicts partial target gene of hsa-miR-302a/b/c/d-3p and hsa-miR-24-3p"

miRNA 预测靶基因 miRNA与靶基因结合位点
预测的目标区域（顶部）和miRNA（底部）的配对情况
（下划线区域为种子区）		 上下游配对
百分数/%
hsa-miR-302a/b/c/d-3p FGF19 5’...UAGAACCCUUUCCCCAGCACUUG...
3’ AGUGGUUUUGUACCUUCGUGAAU		 91
EGFR 5’...GAAAAGCAAUAACAU- - -AGCACUUU
3’ AGUGGUUUUGUACCUUCGUGAAU		 87
hsa-miR-24-3p NEUROD2 5’...UGCAAGGAGGCUCCACUGAGCCU...
3’ GACAAGGACGACUUGACUCGGU		 74
NEUROD1 5’...AUUCAUGUAAUAAAUCUGAGCCU...
3’ GACAAGGACGACUUGACUCGGU		 96
NET1 5’...AAGCCAUACUGUUUU--UGAGCCAA...
3’ GACAAGGACGACUUGACUCGGU		 89
NREP 5’ ...AGAAACUAUGGGACUCUGAGCCU...
3’ GACAAGGACGACUUGACUCGGU		 98
FGF11 5’...CCCUUUUCAUUGCCA--CUGAGCCA...
3’ GACAAGGACGACUUGACUCGGU		 99
FGFBP3 5’...UGAGCAAUUGCCAACCUGAGCCA...
3’ GACAAGGACGACUUGACUCGGU		 97
FGFR3 5’...CUCCCACACCCAAAGCUGAGCCU...
3’ GACAAGGACGACUUGACUCGGU		 88
PDGFRB 5’...GGAGGCCAACUGACU--CUGAGCCA...
3’ GACAAGGACGACUUGACUCGGU		 97
PDGFRA 5’...UAGUAAGUGCGAAGACUGAGCCA...
3’ GACAAGGACGACUU-GACUCGGU		 95
ANGPT4 5’...GACACCCUGGGCUUCCUGAGCCA...
3’ GACAAGGACGACUU-GACUCGGU		 96
IGFBP5 5’...UCCCCUGAGAAAAGACUGAGCCA...
3’GACAAGGACGACUU---GACUCGGU		 95

Fig 4

GO annotation classification of target genes of differential miRNA in 2D and 3D-DPSC-Exo"

Fig 5

GO term histogram of enrichment of target genes of differential miRNA in biological process, cell component and molecular function of 2D-DPSC-Exo and 3D-DPSC-Exo (Qvalue≤0.05)"

Fig 6

KEGG enrichment pathway of target genes of differential miRNA in 2D-DPSC-Exo and 3D-DPSC-Exo (Qvalue≤0.05)"

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