CircPRKD3 Promotes Osteogenic Differentiation Of Periodontal Ligament Stem Cells Under Mechanical Force By Sponging MiR-6783-3p

BackgroundOrthodontic treatment can treat malocclusion malocclusion so that the structure and shape of the teeth,maxillocraniofacial surface of patients can achieve balance and coordination,but the efficient treatment mainly depends on the precise control of the movement of the teeth.The biological basis of orthodontic tooth movement(OTM)is the resorption and formation of alveolar bone to complete the reconstruction in dynamic balance.The bone remodeling microenvironment is regulated by a complex array of periodontal ligament stem cells(PDLSCs),whose core is the stressed tooth,senses mechanical stress and transforms it into biochemical signals,activating intracellular cascade signals and triggering cellular biochemical reactions.Thus osteogenic differentiation and regulation of periodontal tissue remodeling,alveolar bone tissue remodeling and tooth movement.A large number of studies have confirmed that noncoding RNA(ncRNA)can be involved in the regulation of mechanically induced bone remodeling of PDLSCs,circular Rnas(Circrnas)are specially closed circular Ncrnas formed as covalent bonds and play an important regulatory role in a variety of biological processes.Our research group and many previous studies confirmed that miR-21 can be involved in regulating PDLSCs bone remodeling in OTM through various ways.Based on the competitive endogenous RNA(ceRNA)theory,we further determine circPRKD3,which is related to miR-21 and has the most obvious endogenous expression difference under mechanical induction.However,the response of PDLSCs to mechanical stimulation and their involvement in regulating osteogenic differentiation under mechanical forces remain unclear.Objectives(1)To explore the expression of circPRKD3 of PDLSCs under mechanical force.(2)To explore the effect of circPRKD3 on the osteogenic differentiation of PDLSCs under mechanical force.(3)To screen and validate the downstream targets of circPRKD3 and explore the mechanism of its involvement in regulating osteogenic differentiation of PDLSCs under mechanical force,so as to increase the research basis for elucidating the mechanism of PDLSCs regulating bone remodeling in periodontal tissues in OTM,and to provide new ideas for exploring targeted nanomedicine development for precise regulation of OTM.Materials and methods(1)Human PDLSCs were isolated and cultured in vitro,and the BD Accuri C6 flow cytometer was used to identify the surface markers of PDLSCs,and their multidirectional differentiation potential was identified by Alizarin red/Alkaline phosphatase/O il red O staining after induction of osteogenic/lipogenic differentiation.(2)The cells were induced by Flexcell FX-6000 Strain Unit at 8%amplitude and 0.5 Hz for 24 h.The changes of circPRKD3 expression level were analyzed by qRT-PCR and Western Blot.(3)Construct lentiviral vector/plasmid vector to establish circPRKD3 knockdown and overexpression PDLSCs model using transfection technique and verify its transfection efficiency.After 24 h of induction using mechanical force,at the transcriptional and translational levels,the expression of RUNX2、ALP were assessed to explore the effect of circPRKD3 on the osteogenic differentiation process of PDLSCs under mechanical force.(4)Immunofluorescence test and Nucleocytoplasmic separation test were used to localize the distribution of circPRKD3,and bioinformatics analysis was applied to predict the downstream molecules of circPRKD3,and the most significantly differentially expressed mRNA were screened in the overexpression and silencing models of circPRKD3.DualLuciferase Reporter Assay was applied to further validate the interaction of circPRKD3 and miR-6783-3p.MiR-6783-3P mimics was used to enhance the endogenous expression of miR6783-3p and detect its regulatory effect on osteogenic differentiation markers.The interaction of miR-6783-3p with circPRKD3 was verified using cell co-transfection technique.Results(1)The PDLSCs express MSC surface markers CD73,CD90,CD146 and STRO-1 and have good osteogenic/lipogenic differentiation potential.(2)The expression of circPRKD3 was increased during osteogenic differentiation of PDLSCs under mechanical force.(3)After knockdown of circPRKD3,osteogenic differentiation markers ALP and RUNX2 were decreased at both transcriptional and translational levels.Overexpression of circPRKD3 enhanced the expression of RUNX2 at both transcriptional and translational levels,while ALP was elevated only at transcriptional level.(4)circPRKD3 is mainly distributed in the cytoplasm and can act as a molecular sponge for miR-6783-3p.After increasing the regulatory effect of miR-6783-3p,The transcriptional expression of RUNX2 was enhanced,while the expression of ALP was not significantly changed,and Western Blot confirmed that the protein expression levels of both were elevated.The co-transfection results showed that the expression of osteogenic differentiation marker proteins ALP and RUNX2 was decreased in the miR-6783-3p and circPRKD3 co-transfection group compared with the miR-6783-3p/circPRKD3 transfection group.Conclusion(1)Mechanical force increased the expression of circPRKD3 of PDLSCs.(2)Knockdown of circPRKD3 hindered PDLSCs from osteogenesis under mechanical force,while overexpression of circPRKD3 promoted the early osteogenesis process of PDLSCs.The positive regulation of circPRKD3 on the osteogenic differentiation of PDLSCs under mechanical force was found for the first time.(3)The positive role of miR-6783-3p in bone formation was verified for the first time,and it was revealed that circPRKD3 could act as a molecular sponge of miR-6783-3p,exerting competitive endogenous RNA(ceRNA)to regulate the expression of downstream target genes,the mechanism of indirect regulation of alveolar bone formation.