MiR-363-3p attenuates simvastatin-induced osteogenic differentiation of periodontal ligament stem cells by targeting KLF2

Abstract Purpose Simvastatin has been proven to induce osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Besides, simvastatin has been confirmed to affect the progression of many system diseases by regulating miRNA. However, whether miRNA is involved in the osteogenic differentiation of PDLSCs induced by simvastatin has not been reported and needs further study. Methods Human PDLSCs were successfully isolated. Flow cytometry was used to identify PDLSCs. After PDLSCs were treated with simvastatin, mineralized nodules, alkaline phosphatase (ALP), osteogenic-related gene, miR-363-3p, kruppel-like factor 2 (KLF2), and runt-related transcription factor 2 (Runx2) levels were determined by alizarin red staining, enzyme linked immunosorbent assay, quantitative real-time polymerase chain reaction, and western blot. After PDLSCs transfected with or without miR-363-3p mimic were treated with simvastatin, mineralized nodules, ALP, osteogenic-related gene, miR-363-3p, β-catenin, KLF2, and Runx2 levels were tested again. The relationship between miR-363-3 and KLF2 was analyzed by bioinformatics and dual-luciferase assay. KLF2 and Runx2 interaction was detected by Co-Immunoprecipitation. Results PDLSCs were positive for STRO-1 and CD44 but negative for hematopoietic marker (CD34). Simvastatin intensified mineralized nodules, ALP activity, and osteogenic-related genes level and reduced miR-363-3p level, which was reversed by miR-363-3p mimic. Moreover, miR-363-3p targeted KLF2 which interacted with Runx2. We further confirmed that simvastatin elevated β-catenin, KLF2, and Runx2 levels, while the above effects were overturned by miR-363-3p mimic. Conclusion MiR-363-3p restrained the osteogenic differentiation of simvastatin-induced hPDLSCs by repressing KLF2/Runx2 and β-catenin expression.