Li Fang-Qi, Chen Wen-Bo, Luo Zhi-Wen, Chen Yi-Sheng, Sun Ya-Ying, Su Xiao-Ping, Sun Jun-Ming, Chen Shi-Yi
Department of Sports Medicine, Huashan Hospital, Fudan University, Shanghai 200040, China.
Guangxi Key Laboratory of Oral and Maxillofacial Rehabilitation and Reconstruction, Guangxi Medical University, Nanning 530021, Guangxi Zhuang Autonomous Region, China.
World J Stem Cells. 2023 Apr 26;15(4):248-267. doi: 10.4252/wjsc.v15.i4.248.
Fibroblast plays a major role in tendon-bone healing. Exosomes derived from bone marrow mesenchymal stem cells (BMSCs) can activate fibroblasts and promote tendon-bone healing the contained microRNAs (miRNAs). However, the underlying mechanism is not comprehensively understood. Herein, this study aimed to identify overlapped BMSC-derived exosomal miRNAs in three GSE datasets, and to verify their effects as well as mechanisms on fibroblasts.
To identify overlapped BMSC-derived exosomal miRNAs in three GSE datasets and verify their effects as well as mechanisms on fibroblasts.
BMSC-derived exosomal miRNAs data (GSE71241, GSE153752, and GSE85341) were downloaded from the Gene Expression Omnibus (GEO) database. The candidate miRNAs were obtained by the intersection of three data sets. TargetScan was used to predict potential target genes for the candidate miRNAs. Functional and pathway analyses were conducted using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively, by processing data with the Metascape. Highly interconnected genes in the protein-protein interaction (PPI) network were analyzed using Cytoscape software. Bromodeoxyuridine, wound healing assay, collagen contraction assay and the expression of COL I and α-smooth muscle actin positive were applied to investigate the cell proliferation, migration and collagen synthesis. Quantitative real-time reverse transcription polymerase chain reaction was applied to determine the cell fibroblastic, tenogenic, and chondrogenic potential.
Bioinformatics analyses found two BMSC-derived exosomal miRNAs, has-miR-144-3p and has-miR-23b-3p, were overlapped in three GSE datasets. PPI network analysis and functional enrichment analyses in the GO and KEGG databases indicated that both miRNAs regulated the PI3K/Akt signaling pathway by targeting phosphatase and tensin homolog (PTEN). experiments confirmed that miR-144-3p and miR-23b-3p stimulated proliferation, migration and collagen synthesis of NIH3T3 fibroblasts. Interfering with PTEN affected the phosphorylation of Akt and thus activated fibroblasts. Inhibition of PTEN also promoted the fibroblastic, tenogenic, and chondrogenic potential of NIH3T3 fibroblasts.
BMSC-derived exosomes promote fibroblast activation possibly through the PTEN and PI3K/Akt signaling pathways, which may serve as potential targets to further promote tendon-bone healing.
成纤维细胞在肌腱-骨愈合中起主要作用。骨髓间充质干细胞（BMSCs）来源的外泌体可通过所含的微小RNA（miRNAs）激活成纤维细胞并促进肌腱-骨愈合。然而，其潜在机制尚未完全明确。在此，本研究旨在在三个GSE数据集中鉴定BMSCs来源的外泌体miRNAs的重叠部分，并验证它们对成纤维细胞的作用及其机制。
在三个GSE数据集中鉴定BMSCs来源的外泌体miRNAs的重叠部分，并验证它们对成纤维细胞的作用及其机制。
从基因表达综合数据库（GEO）下载BMSCs来源的外泌体miRNAs数据（GSE71241、GSE153752和GSE85341）。通过三个数据集的交集获得候选miRNAs。使用TargetScan预测候选miRNAs的潜在靶基因。分别使用基因本体论（GO）和京都基因与基因组百科全书（KEGG）数据库，通过Metascape处理数据进行功能和通路分析。使用Cytoscape软件分析蛋白质-蛋白质相互作用（PPI）网络中高度相互连接的基因。采用溴脱氧尿苷、伤口愈合试验、胶原收缩试验以及I型胶原和α-平滑肌肌动蛋白阳性的表达来研究细胞增殖、迁移和胶原合成。应用定量实时逆转录聚合酶链反应来确定细胞的成纤维、成腱和成软骨潜能。
生物信息学分析发现两个BMSCs来源的外泌体miRNAs，即has-miR-144-3p和has-miR-23b-3p，在三个GSE数据集中重叠。PPI网络分析以及GO和KEGG数据库中的功能富集分析表明，这两种miRNAs均通过靶向磷酸酶和张力蛋白同源物（PTEN）调节PI3K/Akt信号通路。实验证实，miR-144-3p和miR-23b-3p刺激NIH3T3成纤维细胞的增殖、迁移和胶原合成。干扰PTEN影响Akt的磷酸化，从而激活成纤维细胞。抑制PTEN也促进了NIH3T3成纤维细胞的成纤维、成腱和成软骨潜能。
BMSCs来源的外泌体可能通过PTEN和PI3K/Akt信号通路促进成纤维细胞激活，这可能作为进一步促进肌腱-骨愈合的潜在靶点。
