三维培养牙髓间充质细胞外泌体微小RNA表达谱分析

doi:10.7518/gjkq.2022026

国际口腔医学杂志 ›› 2022, Vol. 49 ›› Issue (1): 27-36.doi: 10.7518/gjkq.2022026

三维培养牙髓间充质细胞外泌体微小RNA表达谱分析

艾晓青(),窦磊,乔新,杨德琴()

重庆医科大学附属口腔医院牙体牙髓科口腔疾病与生物医学重庆市重点实验室重庆市高校市级口腔生物医学工程重点实验室重庆 401147

收稿日期:2021-04-05 修回日期:2021-10-14 出版日期:2022-01-01 发布日期:2022-01-07
通讯作者: 杨德琴
作者简介:艾晓青,硕士,Email： 2018110850@stu.cqmu.edu.cn
基金资助:
国家自然科学基金(31970783);国家自然科学基金青年基金(81800958)

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

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

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
Supported by:
This study was supported by National Natural Science Foundation of China(31970783);National Natural Science Foundation of China Youth Fund(81800958)

摘要/Abstract

摘要：

目的分析并比较二维（2D）与三维（3D）培养条件下人牙髓间充质细胞（DPSCs）外泌体（Exo）微小RNA（miRNA）的表达谱。方法 2D与3D条件下分别培养DPSCs,提取细胞Exo,采用透射电子显微镜、蛋白质免疫印迹及纳米颗粒追踪分析等方法观察并鉴定;通过高通量测序筛选差异表达miRNA,采用Dr.Tom系统及TargetScan网站进行生物信息学分析及组织再生修复相关靶基因预测。结果 3D培养下收集的DPSC-Exo均呈现“茶托样”双层膜结构,CD63和CD9表达阳性,粒径分布符合外泌体特征,与2D培养的DPSC-Exo一致。高通量测序共计检测外泌体来源miRNA 253个,其中3D组表达222个,特有表达99个;与2D组相比,表达显著差异60个[︱log₂（3D/2D）︱≥1,Qvalue≤0.001]。差异miRNA主要参与的生物学过程与分子功能分别为细胞过程和结合;京都基因与基因组数据库（KEGG）通路富集分析显示在代谢通路有显著富集。miR-302的候选靶基因有成纤维细胞生长因子（FGF）19和表皮生长因子受体等;miR-24-3p可能靶向神经元分化因子2、神经上皮细胞转化因子1、神经元分化因子1、神经元再生相关蛋白、FGF11、FGF结合蛋白3、FGF受体3、血小板衍生生长因子受体（PDGFR）β多肽、PDGFRα多肽、血管生成素、胰岛素样生长因子结合蛋白5等,以发挥组织再生修复功能。结论相比2D培养,3D培养能调节DPSC-Exo部分miRNA表达量,上调一些与再生修复相关的miRNA,3D培养可考虑作为调节外泌体应用潜能的一种手段。

关键词: 三维培养, 牙髓间充质细胞, 外泌体测序, 微小RNA

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

艾晓青,窦磊,乔新,杨德琴. 三维培养牙髓间充质细胞外泌体微小RNA表达谱分析[J]. 国际口腔医学杂志, 2022, 49(1): 27-36.

Ai Xiaoqing,Dou Lei,Qiao Xin,Yang Deqin. MicroRNA profile of exosomes derived from dental pulp stromal cells under three-dimensional culture condition[J]. Int J Stomatol, 2022, 49(1): 27-36.

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图4

2D-DPSC-Exo与3D-DPSC-Exo显著差异表达miRNA靶基因的GO注释分类 x轴为GO功能分类,y轴为注释到GO术语（GO Term）上的基因数目。蓝色部分为biological process（生物学过程）,橙色部分为cellular component（细胞组分）,浅蓝色部分为molecular function（分子功能）。生物学过程包括cellular process（细胞过程）、biological regulation（生物调节）、me-tabolic process（代谢过程）、regulation of biological process（生物过程调节）、response to stimulus（对刺激的反应）、multicellular organismal process（多细胞生物过程）、cellular component organization or biogenesis（细胞成分组织或生物发生）、localization（定位）、signaling（信号）、developmental process（发育过程）、positive regulation of biological process（生物过程的正调节）、negative regulation of biological process（生物过程的负调节）、immune system process（免疫系统过程）、multi-organism process（多生物过程）、cell proliferation（细胞增殖）、locomotion（移动）、biological adhesion（生物附着）、reproduction（繁殖）、reproductive process（繁殖过程）、growth（生长）、behavior（行为）、biological_process（生物学过程）、rhythmic process（节律过程）、cell killing（细胞杀伤）、detoxification（排毒）、pigmentation（色素沉着）、cell aggregation（细胞集聚）、biological phase（生物阶段）、nitrogen utilization（氮利用）、carbohydrate utilization（糖利用）。细胞组分包括cell（细胞）、cell part（细胞部分）、organelle（细胞器）、organelle part（细胞器组分）、membrane（膜）、membrane part（膜组分）、protein-containing complex（含蛋白质复合物）、membrane-enclosed lumen（膜封闭腔）、extracellular region（胞外区域）、extracellular region part（胞外区组分）、cell junction（细胞接合）、synapse（突触）、synapse part（突触组分）、supramolecular complex（超分子复合物）、cellular_component（细胞组分）、nucleoid（类核）、other organism part（其他有机体组分）、other organism（其他有机体）。分子功能包括binding（结合）、catalytic activity（催化活性）、transcription regulator activity（转录调节活性）、molecular function regulator（分子功能调节）、molecular transducer activity（分子转导活性）、transporter activity（转运活性）、structural molecule activity（结构分子活性）、molecular_function（分子功能）、cargo receptor activity（转运受体活性）、hijacked molecular function（拦截分子功能）、antioxidant activity（抗氧化活性）、translation regulator activity（翻译调节活性）、molecular carrier activity（分子载体活性）、protein tag（蛋白标记）。"

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图5

2D-DPSC-Exo与3D-DPSC-Exo显著差异表达miRNA靶基因分别在生物学过程、细胞组分和分子功能的GO富集情况（Qvalue≤0.05） x轴为富集比例,y轴为GO术语（GO Term）。图中气泡的大小表示注释到某个GO Term上的差异基因数目（gene number）,颜色越蓝代表显著性值（即Qvalue值）越小,富集程度越高。A为biological process（生物学过程）,B为cellular component（细胞组分）,C为molecular function（分子功能）。生物学过程包括regulation of transcription by RNA polymerase Ⅱ （RNA聚合酶Ⅱ对转录的调控）、phosphorylation（磷酸化）、ion transport（离子转运）、immune system process（免疫系统过程）、lipid metabolic process（脂质代谢过程）、transmembrane transport（跨膜转运）、multicellular organism development（多细胞生物发育）、cell differentiation（细胞分化）、biological_process（生物学过程）、oxidation-reduction process（氧化还原过程）、proteolysis（蛋白酶解）、cell cycle（细胞周期）、cell projection organization（组织细胞投影）、positive regulation of transcription by RNA polymerase Ⅱ（RNA聚合酶Ⅱ对转录的正调控）、mRNA processing（mRNA加工）、cell adhesion（细胞黏附）、peptidyl-tyrosine phosphorylation（肽基酪氨酸磷酸化）、protein transport（蛋白质转运）、ion transmembrane transport（离子跨膜运输）、positive regulation of nucleic acid-templated transcription（核酸模板转录的正调控）。细胞组分包括membrane（膜）、cell projection（细胞投影）、cytoskeleton（细胞骨架）、cytoplasm（细胞质）、cytoplasmic vesicle（胞质囊泡）、endoplasmic reticulum（内质网）、endosome（核内体）、golgi apparatus（高尔基体）、mitochondrion（线粒体）、cell junction（细胞连接）、synapse（突触）、integral component of plasma membrane（质膜的组成部分）、cellular_component（细胞组分）、microtubule organizing center （微管组织中心）、intracellular membrane-bounded organelle（细胞内膜结合的细胞器）、cilium（鞭毛）、nucleoplasm（核质）、plasma membrane（质膜）、neuron projection（神经元投射）、integral component of membrane（膜的组成部分）。分子功能包括protein binding（蛋白结合）、transferase activity（转移酶活性）、hydrolase activity（水解酶活性）、nucleic acid binding（核酸结合）、kinase activity（激酶活性）、nucleotide binding（核苷酸结合）、catalytic activity（催化活性）、oxidoreductase activity（氧化还原酶活性）、molecular_function（分子功能）、peptidase activity（肽酶活性）、DNA-binding transcription activator activity, RNA polymerase Ⅱ-specific （DNA结合转录激活剂活性RNA聚合酶Ⅱ特异性）、protein kinase activity（蛋白激酶活性）、sequence-specific DNA binding（序列特异性 DNA 结合）、lyase activity（裂解酶活性）、ion channel activity（离子通道活性）、transferase activity, transferring acyl groups[转移酶活性（转移酰基）]、transferase activity, transferring glycosly groups[转移酶活性（转移糖基）]、lipid binding（脂质结合）、isomerase activity（异构酶活性）、nuclease activity（核酸酶活性）。"

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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

miRNA	预测靶基因	miRNA与靶基因结合位点
miRNA	预测靶基因	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

三维培养牙髓间充质细胞外泌体微小RNA表达谱分析

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

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