Fujii Yasuyuki, Minami Sakura, Hatori Ayano, Kawase-Koga Yoko, Ogasawara Toru, Chikazu Daichi
Department of Oral and Maxillofacial Surgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, Tokyo 160-0023, Japan.
Department of Oral and Maxillofacial Surgery, School of Medicine, Tokyo Women's Medical University, 8-1 Kawadachou, Shinjuku-ku, Tokyo 160-0023, Japan.
Curr Issues Mol Biol. 2024 Sep 28;46(10):10960-10968. doi: 10.3390/cimb46100651.
Dental pulp stem cells (DPSCs) demonstrate high proliferative and multilineage differentiation potential. As previously reported, the helioxanthin derivative 4-(4-methoxyphenyl)pyrido[40,30:4,5]thieno[2,3-b]pyridine-2-carboxamide (TH) has been demonstrated to induce the osteogenic differentiation of DPSCs. However, the mechanism of osteogenesis induced by TH in DPSCs remains unknown. The objective of this study was to identify functional extracellular vesicle (EV) microRNAs (miRNAs), and the principal genes involved in the TH-induced osteogenesis of DPSCs. DPSCs were derived from dental pulp extracted from the third molars of three healthy subjects, and were cultured with or without TH. miRNAs were extracted from DPSC-derived EVs. The gene expression patterns of mRNA and miRNA were compared using RNA-Seq and miRNA-Seq. To investigate miRNA/mRNA interacting networks, functional analyses were performed by Ingenuity Pathway Analysis. Alkaline phosphatase (ALP) staining demonstrated that treatment with TH resulted in enhanced ALP activity in DPSCs after 7 days. The expression levels of ALP and type 1 collagen alpha 1 were significantly higher in TH-induced DPSCs on day 7. RNA-Seq and miRNA-Seq analyses identified 869 differentially expressed genes (DEGs) and 18 miRNA-DEGs. Gene Ontology analysis of the mRNA-Seq results showed that TH induced several biological activities associated with signal transduction, cell adhesion, and cell differentiation. Integrated miRNA-mRNA analyses showed that these miRNAs contain the targeting information of 277 mRNAs of the DEGs. Among them, 17 target genes known to be involved in the differentiation of osteoblasts, and 24 target genes known to be involved in the differentiation of bone cells were identified. Quantitative real-time PCR showed that expression in DPSCs was upregulated by 48 h of TH treatment. Upstream regulator analysis indicated that , , and may be responsible for gene expression changes in DPSCs after TH treatment. EV miRNA regulatory networks might play crucial roles in TH-induced osteogenic differentiation of DPSCs. Our results presented herein offer valuable insights that will facilitate further research into the mechanism of osteogenesis of DPSCs, which is expected to lead to the clinical application of TH-induced DPSCs for bone regeneration. Furthermore, EVs derived from TH-induced DPSCs might be useful as therapeutic tools for bone defects.
牙髓干细胞（DPSCs）具有高增殖能力和多向分化潜能。如先前报道，己证明氦黄质衍生物4-（4-甲氧基苯基）吡啶并[4′,3′:4,5]噻吩并[2,3-b]吡啶-2-甲酰胺（TH）可诱导DPSCs向成骨细胞分化。然而，TH诱导DPSCs成骨的机制仍不清楚。本研究的目的是鉴定功能性细胞外囊泡（EV）微小RNA（miRNA）以及参与TH诱导DPSCs成骨的主要基因。DPSCs取自三名健康受试者第三磨牙的牙髓，并在有或无TH的条件下培养。从DPSC来源的EV中提取miRNA。使用RNA测序（RNA-Seq）和miRNA测序（miRNA-Seq）比较mRNA和miRNA的基因表达模式。为了研究miRNA/mRNA相互作用网络，通过 Ingenuity Pathway Analysis进行功能分析。碱性磷酸酶（ALP）染色显示，TH处理7天后DPSCs中的ALP活性增强。在第7天，TH诱导的DPSCs中ALP和I型胶原α1的表达水平显著更高。RNA-Seq和miRNA-Seq分析鉴定出869个差异表达基因（DEG）和18个miRNA-DEG。对mRNA-Seq结果的基因本体分析表明，TH诱导了与信号转导、细胞黏附及细胞分化相关的多种生物学活性。综合miRNA-mRNA分析表明，这些miRNA包含277个DEG的mRNA的靶向信息。其中，鉴定出17个已知参与成骨细胞分化的靶基因和24个已知参与骨细胞分化的靶基因。定量实时PCR显示，TH处理48小时后DPSCs中的表达上调。上游调节因子分析表明， 、 和 可能是TH处理后DPSCs基因表达变化的原因。EV miRNA调控网络可能在TH诱导的DPSCs成骨分化中起关键作用。我们在此呈现的结果提供了有价值的见解，将有助于进一步研究DPSCs的成骨机制，有望推动TH诱导的DPSCs在骨再生方面的临床应用。此外，TH诱导的DPSCs来源的EV可能作为治疗骨缺损的工具。
