Lineage-specific exosomes promote the odontogenic differentiation of human dental pulp stem cells (DPSCs) through TGF1/smads signaling pathway via transfer of microRNAs
Lineage-specific exosomes promote the odontogenic differentiation of human dental pulp stem cells (DPSCs) through TGF1/smads signaling pathway via transfer of microRNAs
复制标题
谱系特异性外泌体通过 TGFβ1/smads 信号通路通过 microRNA 的转移促进人牙髓干细胞 (DPSC) 的牙源性分化
DOI：
10.1186/s13287-019-1278-x
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发表时间：
2019-06-13
影响因子：
7.5
通讯作者：
Zheng, Jianmao
中科院分区：
文献类型：
--
作者：
Hu, Xiaoli;Zhong, Yingqun;Zheng, Jianmao
背景牙髓干细胞（dental pulp stem cells，DPSCs）来源的exosomes可作为仿生工具诱导干细胞向牙源性分化，但其微RNA的调控机制和功能尚不清楚。本研究旨在阐明人DPSCs来源的exosomes中所含的microRNA及其潜在的信号级联在牙向分化中的作用.MethodsExosomes从人DPSCs中分离培养，分别命名为UN-Exo和OD-Exo。进行microRNA测序以探索UN-Exo和OD-Exo中包含的microRNA谱。进行通路分析以检测与预测的microRNA靶基因相关的富集通路。通过抑制或过表达（miRNA抑制剂和miRNA模拟物）研究了OD-Exo中高度表达的microRNA的调节作用。采用免疫印迹法检测外泌体microRNA的功能及TGFβ1/smads信号通路在DPSCs牙向分化中的作用。结果OD-Exo的内吞作用通过上调DSP、miR-1、ALP和RUNX 2蛋白的表达，促进DPSCs向牙源性分化。microRNA测序结果显示，OD-Exo中有28个microRNA发生了显著变化，其中7个增加，21个减少。通路分析显示，差异表达microRNA靶向的基因参与多种信号转导，包括TGFβ通路。15个差异表达的microRNA靶向的16个基因参与TGFβ信号转导。Western blot结果显示，OD-Exo通过上调DPSCs中TGFβ1、TGF β 1、p-Smad 2/3和Smad 4的表达，激活TGFβ1通路。因此，一旦TGFβ1信号通路被SB 525334抑制，用OD-Exo处理的DPSC中p-Smad 2/3、DSP和TGF β 1的蛋白水平显著降低。miR-27 a-5 p在OD-Exo中的表达是OD-Exo的11倍，miR-27 a-5 p可促进DPSCs的牙向分化，并通过下调抑制分子LTBP 1，显著上调TGFβ1、TGF β 1、p-Smad 2/3和Smad 4的表达。牙源性条件下分离的OD-Exo是较好的DPSC分化诱导剂。ExosomemicroRNAs通过下调LTBP 1，通过TGFβ1/smads信号通路促进牙向分化。
BackgroundExosomes derived from dental pulp stem cells (DPSCs) can be used as biomimetic tools to induce odontogenic differentiation of stem cells, but the regulatory mechanisms and functions of exosome-encapsulated microRNAs are still unknown. The present study aimed to clarify the role of microRNAs contained in the exosomes derived from human DPSCs and their potential signaling cascade in odontogenic differentiation.MethodsExosomes were isolated from human DPSCs cultured undergrowth and odontogenic differentiation conditions, named UN-Exo and OD-Exo, respectively. The microRNA sequencing was performed to explore the microRNA profile contained in UN-Exo and OD-Exo. Pathway analysis was taken to detect enriched pathways associated with the predicted target genes of microRNAs. The regulatory roles of a highly expressed microRNA in OD-Exo were investigated through its inhibition or overexpression (miRNA inhibitors and miRNA mimics). Automated western blot was used to identify the function of exosomal microRNA and the roles of TGFβ1/smads pathway in odontogenic differentiation of DPSCs. A luciferase reporter gene assay was used to verify the direct target gene of exosomal miR-27a-5p.ResultsEndocytosis of OD-Exo triggered odontogenic differentiation of DPSCs by upregulating DSP, DMP-1, ALP, and RUNX2 proteins. MicroRNA sequencing showed that 28 microRNAs significantly changed in OD-Exo, of which 7 increased and 21 decreased. Pathway analysis showed genes targeted by differentially expressed microRNAs were involved in multiple signal transductions, including TGFβ pathway. 16 genes targeted by 15 differentially expressed microRNAs were involved in TGFβ signaling. Consistently, automated western blot found that OD-Exo activated TGFβ1 pathway by upregulating TGFβ1, TGFR1, p-Smad2/3, and Smad4 in DPSCs. Accordingly, once the TGFβ1 signaling pathway was inhibited by SB525334, protein levels of p-Smad2/3, DSP, and DMP-1 were significantly decreased in DPSCs treated with OD-Exo. MiR-27a-5p was expressed 11 times higher in OD-Exo, while miR-27a-5p promoted odontogenic differentiation of DPSCs and significantly upregulated TGFβ1, TGFR1, p-Smad2/3, and Smad4 by downregulating the inhibitory molecule LTBP1.ConclusionsThe microRNA expression profiles of exosomes derived from DPSCs were identified. OD-Exo isolated under odontogenic conditions were better inducers of DPSC differentiation. Exosomal microRNAs promoted odontogenic differentiation via TGFβ1/smads signaling pathway by downregulating LTBP1.