PWAR6 interacts with miR-106a-5p to regulate the osteogenic differentiation of human periodontal ligament stem cells

Abstract

Human periodontal ligament stem cells (hPDLSCs) associated with bone regeneration serve an important role in the treatment of periodontal disease. Long non-coding RNAs are involved in the osteogenesis of multiple stem cells and can act as a sponge of microRNAs (miRs). The present study aimed to investigate the interaction between Prader Willi/Angelman region RNA 6 (PWAR6) and miR-106a-5p, as well as their influences on the osteogenic differentiation of hPDLSCs. hPDLSCs were isolated and cultured in osteogenic medium (OM) or growth medium (GM) for 7 days prior to transfection with PWAR6overexpression vector, short hairpin RNA PWAR6 or miR-106a-5p mimic. The expression levels of runt-related transcription factor 2, osteocalcin and bone morphogenetic protein 2 (BMP2) were detected by western blotting and reverse transcription-quantitative PCR (RT-qPCR), and the expression levels of PWAR6, miR-106a-5p and alkaline phos- phatase (ALP) were determined by RT-qPCR. ALP activity assays and Alizarin red staining were performed to detect osteogenesis and mineralization, respectively. Luciferase activities of wild-type and mutant PWAR6 and BMP2 were assessed by conducting a dual-luciferase reporter assay. The results indicated that PWAR6 expression was upregulated in OM-incubated hPDLSCs compared with GM-incubated hPDLSCs, and PWAR6 overexpression increased the osteogenic differentiation and mineralization of hPDLSCs compared with the corresponding control group. By contrast, miR-106a-5p expression was downregulated in OM-incubated hPDLSCs compared with GM-incubated hPDLSCs. PWAR6 acted as a sponge of miR-106a-5p and PWAR6 overexpression promoted the osteogenesis of miR-106a-5p mimic-trans- fected hPDLSCs. BMP2 was predicted as a target gene of miR-106a-5p. Collectively, the results indicated that PWAR6 displayed a positive influence on the osteogenic differentiation of hPDLSCs. The results of the present study demonstrated that the PWAR6/miR-106a-5p interaction network may serve as a potential regulatory mechanism underlying hPDLSCs osteogenesis.