Fang Fuchun, Guo Xiaolan, Liu Sitong, Dang Longrui, Chen Zehao, Yang Yumeng, Chen Lu, Lin Jiahao, Qiu Wei, Chen Zhao, Wu Buling
Department of Stomatology, Nanfang Hospital, Southern Medical University, Guangzhou, China.
Shenzhen Stomatology Hospital (Pingshan), Southern Medical University, Shenzhen, China.
Stem Cell Res Ther. 2025 Mar 26;16(1):149. doi: 10.1186/s13287-025-04274-w.
Alternative splicing not only expands the genetic encoding of genes but also determines cellular activities. This study aimed to elucidate the regulation mechanism and biological functions of lincRNA-ASAO in the process of odontogenesis-related genes alternative splicing mediated odontogenic differentiation of hDPSCs.
RACE, RNA-seq, FISH and bioinformatics techniques were used to identify novel lincRNA-ASAO. ALP staining, alizarin red staining, qRT-PCR and western blot were used to identify the role of lincRNA-ASAO in regulating the odontoblast differentiation of hDPSCs. The binding protein PTBP1 of lincRNA-ASAO was screened by RNA-Pulldown, protein profiling and bioinformatics. The target gene ALPL of lincRNA-ASAO/PTBP1 was identified by RNA-seq, bioinformatics technology and DNA agarose gel electrophoresis. FISH, IF, PAR-CLIP and bioinformatics techniques were used to determine the roles of lincRNA-ASAO, PTBP1 and ALPL pre-mRNA in the odontoblast differentiation of hDPSCs.
We identified a novel lincRNA-ASAO that could promote the odontogenic differentiation of human Dental Pulp Stem Cells (hDPSCs). And, the interaction between lincRNA-ASAO and alternative splicing factor PTBP1 promoted the odontoblast differentiation of hDPSCs. In addition, lincRNA-ASAO forms duplexes with ALPL pre-mRNA, targeting PTBP1 to exonic splicing silencer (ESS) of ALPL and regulating exon 2 skipping. Notably, lincRNA-ASAO/PTBP1 regulated ALPL production to increase the type 2 splice variant, which promoted the odontoblast differentiation of hDPSCs.
We have identified the novel lincRNA-ASAO, which can promote the odontoblast differentiation of hDPSCs. The mechanism study found that lincRNA-ASAO/PTBP1 mediated the exon 2 skipping of ALPL pre-mRNA, resulting in the type 2 splice variant of ALPL. Our results enrich the understanding of lncRNAs and alternative splicing in regulating the odontoblast differentiation of hDPSCs, and provide clues to improve the clinical therapeutic potential of hDPSCs for dental pulp restoration.
可变剪接不仅扩展了基因的遗传编码，还决定了细胞活性。本研究旨在阐明长链非编码RNA-ASAO（lincRNA-ASAO）在牙源性相关基因可变剪接介导的人牙髓干细胞（hDPSCs）成牙分化过程中的调控机制及生物学功能。
采用RACE、RNA测序、荧光原位杂交（FISH）和生物信息学技术鉴定新型lincRNA-ASAO。采用碱性磷酸酶（ALP）染色、茜素红染色、qRT-PCR和蛋白质免疫印迹法鉴定lincRNA-ASAO在调控hDPSCs成牙本质细胞分化中的作用。通过RNA下拉、蛋白质谱分析和生物信息学筛选lincRNA-ASAO的结合蛋白PTBP1。通过RNA测序、生物信息学技术和DNA琼脂糖凝胶电泳鉴定lincRNA-ASAO/PTBP1的靶基因碱性磷酸酶基因（ALPL）。采用FISH、免疫荧光（IF）、紫外交联免疫沉淀（PAR-CLIP）和生物信息学技术确定lincRNA-ASAO、PTBP1和ALPL前体mRNA在hDPSCs成牙本质细胞分化中的作用。
我们鉴定出一种新型lincRNA-ASAO，其可促进人牙髓干细胞的成牙分化。并且，lincRNA-ASAO与可变剪接因子PTBP1之间的相互作用促进了hDPSCs的成牙本质细胞分化。此外，lincRNA-ASAO与ALPL前体mRNA形成双链体，将PTBP1靶向ALPL的外显子剪接沉默子（ESS）并调节外显子2跳跃。值得注意的是，lincRNA-ASAO/PTBP1调节ALPL的产生以增加2型剪接变体，从而促进hDPSCs的成牙本质细胞分化。
我们鉴定出新型lincRNA-ASAO，其可促进hDPSCs的成牙本质细胞分化。机制研究发现，lincRNA-ASAO/PTBP1介导ALPL前体mRNA的外显子2跳跃，导致ALPL的2型剪接变体产生。我们的结果丰富了对lncRNAs和可变剪接在调控hDPSCs成牙本质细胞分化中的理解，并为提高hDPSCs用于牙髓修复的临床治疗潜力提供了线索。
