Exosomal circ_0003057 promotes osteo/odontogenic differentiation of hDPSCs by binding with EIF4A3 through upregulated parental gene ANKH

IF 7.1 1区医学 Q1 DENTISTRY, ORAL SURGERY & MEDICINE

International endodontic journal Pub Date : 2025-06-03 DOI:10.1111/iej.14262

Bingtao Wang, Yuanyuan Kong, Huixian Dong, Feng Lai, Zixin Guo, Liecong Lin, Jingyi Xu, Jingkun Zhang, Yiguo Jiang, Qianzhou Jiang

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引用次数: 0

Abstract

Aim

Elucidating the mechanism of osteo/odontogenic differentiation of human dental pulp stem cells (hDPSCs) is crucial for advancing regenerative endodontic procedures (REPs). Circular RNAs (circRNAs) play significant regulatory roles in stem cell differentiation, and exosomes are crucial for intercellular communication. This study investigated the role of exosomal circRNAs in hDPSCs during osteo/odontogenic differentiation using in vitro and in vivo evidence.

Materials and Methods

We isolated hDPSCs from dental pulp tissues of healthy immature permanent teeth. CircRNA microarray analysis was used to identify differentially expressed circRNAs. Exosomes were extracted from hDPSCs using ultracentrifugation, and circRNA content was detected. Functional validation of exosomal circRNAs was performed using siRNA/overexpression plasmids and subcutaneous transplantation in to nude mice. The biological effects of circ_0003057, EIF4A3 and ANKH were determined using real-time quantitative polymerase chain reaction (qRT-PCR), western blotting (WB), alkaline phosphatase (ALP) staining and activity, alizarin red staining (ARS), quantification and immunofluorescence staining. EIF4A3 was identified as a potential binding protein (RBP) for circ_0003057 in the database, and this binding relationship was confirmed using RNA pull-down and RIP assays. qRT-PCR and WB were performed to determine whether the host gene ANKH of circ_0003057 was activated.

Results

circ_0003057 expression was increased during osteo/odontogenic differentiation of hDPSCs, whereas circ_0003057 downregulation suppressed this process. EIF4A3 was confirmed to be a binding protein of circ_0003057 and was upregulated during osteo/odontogenic differentiation of hDPSCs. Further investigation revealed that circ_0003057 upregulation during osteo/odontogenic differentiation led to the upregulation of its parental gene, ANKH. Co-transfection experiments confirmed that circ_0003057 upregulated ANKH, promoting osteo/odontogenic differentiation of hDPSCs.

Conclusions

This study demonstrates that exosomal circ_0003057 promotes osteo/odontogenic differentiation of hDPSCs by interacting with EIF4A3 and upregulating ANKH, providing new insights into the molecular mechanisms underlying this process and its potential applications in regenerative endodontics.

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外泌体circ_0003057通过上调亲本基因ANKH与EIF4A3结合，促进hdpsc的成骨/牙源性分化。

目的：阐明人牙髓干细胞（hDPSCs）成骨/成牙分化的机制对推进再生牙髓治疗（REPs）至关重要。环状rna （circRNAs）在干细胞分化中起着重要的调节作用，外泌体在细胞间通讯中起着至关重要的作用。本研究利用体外和体内证据研究了外泌体环状rna在hdpsc成骨/牙源性分化过程中的作用。材料和方法：从健康未成熟恒牙的牙髓组织中分离出hdpsc。CircRNA微阵列分析用于鉴定差异表达的CircRNA。利用超离心技术从hDPSCs中提取外泌体，检测circRNA含量。使用siRNA/过表达质粒和裸鼠皮下移植对外泌体环状rna进行功能验证。采用实时定量聚合酶链反应（qRT-PCR）、western blotting （WB）、碱性磷酸酶（ALP）染色及活性、茜素红染色（ARS）、定量和免疫荧光染色检测circ_0003057、EIF4A3和ANKH的生物学效应。EIF4A3在数据库中被鉴定为circ_0003057的潜在结合蛋白（RBP），并通过RNA pull-down和RIP实验证实了这种结合关系。采用qRT-PCR和WB检测circ_0003057宿主基因ANKH是否被激活。结果：circ_0003057在hdpsc成骨/牙源性分化过程中表达增加，而circ_0003057下调抑制这一过程。EIF4A3被证实是circ_0003057的结合蛋白，并在hdpsc的成骨/牙源性分化过程中上调。进一步的研究表明，circ_0003057在成骨/牙源性分化过程中的上调导致其亲本基因ANKH的上调。共转染实验证实circ_0003057上调ANKH，促进hdpsc的成骨/牙源性分化。结论：本研究表明外显体circ_0003057通过与EIF4A3相互作用和上调ANKH促进hdpsc的成骨/牙源性分化，为这一过程的分子机制及其在再生牙髓学中的潜在应用提供了新的见解。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

International endodontic journal 医学-牙科与口腔外科

CiteScore

10.20

自引率

28.00%

发文量

195

审稿时长

4-8 weeks

期刊介绍： The International Endodontic Journal is published monthly and strives to publish original articles of the highest quality to disseminate scientific and clinical knowledge; all manuscripts are subjected to peer review. Original scientific articles are published in the areas of biomedical science, applied materials science, bioengineering, epidemiology and social science relevant to endodontic disease and its management, and to the restoration of root-treated teeth. In addition, review articles, reports of clinical cases, book reviews, summaries and abstracts of scientific meetings and news items are accepted. The International Endodontic Journal is essential reading for general dental practitioners, specialist endodontists, research, scientists and dental teachers.