LincRNA-ASAO promotes dental pulp repair through interacting with PTBP1 to increase ALPL alternative splicing

Abstract

Background 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. Methods 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-Pull-down, 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. Results 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. Conclusions 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.

1. Introduction

Eukaryotic genes consist of a combination of exons and introns, and alternative splicing at various sites expands the coding capacity of eukaryotic genes [1]. Peptides generated from a single gene through alternative splicing are often similar but not identical, leading to subtle or significant variations at the mRNA and protein levels [2, 3]. More than 95% of human exonic genes undergo alternative splicing, which is tightly controlled by the interaction between post-transcriptionally acting proteins known as splicing factors and cis-acting nucleotide sequences [4, 5]. These splicing factors encompass members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which either promote or inhibit specific splicing events by interacting with exonic or intronic regulatory sequences categorized as enhancers or silencers [6–9]. Alternative splicing events play a regulatory role in a wide array of cellular biological processes, influencing cell differentiation, proliferation, and apoptosis [10–13].

Stem cells hold immense potential for treating various human diseases and conditions. The process of stem cell differentiation produces multiple transcripts through alternative splicing, generating tissue-specific mRNA and protein isoforms that serve as key regulators of gene expression [14, 15]. Early genome-wide analyses have revealed more than 1000 genes with alternative splicing events in stem cells [16]. Using alternative splicing microarrays, researchers have identified altered splicing patterns in 170 of 40,000 putative exon-exon junctions in embryonic stem cells (ESCs), with 67% of these events predicted to alter protein sequence and domain composition [17]. In recent years, with the successful isolation and culture of hDPSCs, an increasing number of studies have confirmed their important role in pulp injury repair, particularly through differentiation into various cell types to promote pulp tissue regeneration [18]. hDPSCs can differentiate into odontoblas