Abstract. Periodontal ligament-associated protein-1 (PLAP-1/ asporin) is a special marker in periodontal ligament tissue. It is an important regulator of osteogenic differentiation of periodontal ligament cells (PDLCs). This marker is also a prerequisite for periodontal ligament development and mineralization in maintaining homeostasis of the periodontium. However, the molecular mechanisms of the regulation of PLAP-1 expression at the post-transcriptional level remain unknown. By contrast, microRNAs (miRNAs) provide an additional level of regulation beyond that of transcription factors via regulation of the post-transcriptional control of gene expression. This study was designed to analyze miRNA differential expression patterns of PDLCs at various osteoblastic differentiation stages and to determine the contribution of miRNAs in the regulation of PLAP-1 expression during osteoblast differentiation. Bioinformatic analysis was performed to predict miRNAs that potentially regulate the gene expression of PLAP-1. Dual luciferase reporter assay and qRT-PCR were performed to confirm the effects of these miRNAs on PLAP-1 gene expression. Our results indicated that mir-101 and mir-21 target PLAP-1 to regulate its expression during osteogenic differentiation of PDLCs.
IntroductionPeriodontal ligament (PDL), the most important tissue influencing the lifespan of the human tooth, is composed of heterogeneous cell populations including osteoblasts, which have osteogenic differentiation potential (1-3). Osteogenesis of periodontal ligament cells (PDLCs) is part of an important process of cytodifferentiation and mineralization to form mineralized tissue cells (4). Osteoblast differentiation results in the expression of tissue-specific gene products that form and regulate the structure of the mineralized PDL extracellular matrix (5).Periodontal ligament associated protein-1 (PLAP-1/ asporin), a specific marker of the PDL, is an important negative regulator of osteogenic differentiation in maintaining the homeostasis of periodontium (6-8). PLAP-1 was first identified as a novel member of the small leucine-rich repeat proteoglycan (SLRP) family and confirmed to be expressed specifically and predominantly in the PDL (9-10). PLAP-1 was found to be closely associated with the process of cytodifferentiation of PDL cells (11). Therefore, the relative expression level of PLAP-1 may be regarded as a specific marker for the osteogenic differentiation of PDLCs.Moreover, PLAP-1 was not only expressed specifically in the PDL of an erupted tooth but also in the dental follicle, which is the progenitor tissue of the PDL during tooth development (8). This result suggests that PLAP-1 may be involved in PDL development and cytodifferentiation (11).Furthermore, it is important to understand the processes that regulate the mineralization of PDLCs. Previous studies showed that the osteogenesis of PDLCs, a complex process in which multiple signaling pathways are involved, is regulated by numerous growth and transcription factors (12-14). However, the...