Study On The Role Of MiR-195-5p In Orthodontic Tension Force-induced Bone Formation

l.orthodontic tension stress stimulates osteogenesis of periodontal ligament cellsObjectives:Orthodontic tension stress-induced bone formation is a complex biological process.Periodontal ligament cells(PDLCs)showed osteogenesis under orthodontic tension stress and involved in periodontal bone remodeling.The aim of this study was to apply tension stress on PDLCs in vitro and to observe the osteogenic differentiation.Methods:Primary hPDLCs was isolated and cultured.PDLCs were subjected to different time of cyclic tension stress(CTS).Cell morphology,cell arrangement and genes expression were analysed.Western blot was also used to detect osteogenic differentiation markers,Alizarin red staining was used to detect the formation of mineralized nodules under induced conditions.Results:Cell morphology was elongated and the cells were re-arranged by tension stress.The expression levels of ALP and OCN in PDLCs were significantly up-regulated with the increase of the CTS duration.72h CTS applying promoted the accumulation of mineral nodules and the expression of osteogenesis-related genes expression.Conclusions:The experimental CTS model with 12%deformation rate and 6 cycles/min frequency can induce the osteogenesis of PDLCs by 72h.2.Study of miRNA expression in CTS induced PDLCs osteogenesisObjectives:Mechanotranscution and the role of miRNA in orthodontic tension stress induced PDLCs osteogenesis is still unclear.The aim of this study was to investigate the changes of miRNA expression in PDLCs induced by CTS and to explore the relationship between miRNAs and targets in CTS induced osteogenesis.Methods:Primary hPDLCs were isolated and cultured.hPDLCs were subjected to CTS,and miRNA microarray and mRNA microarray were used to detect the miRNA expression profile and mRNA expression profile of CTS stimulated PDLCs.Experimental informatic methods were used to find the core miRNAs and predict their functions.Real-time RT-PCR was used to verify the reliability of these results.Results:In microarray results,compared to unloaded cells,32 miRNAs and over 1000 mRNAs showed significant differences in CTS stimulated PDLCs.Seven core miRNAs were obtained by bioinformatic analysis and 26 of their targets were found to be related to osteogenic differentiation.Real-time RT-PCR showed consistent results to microarray.Conclusions:The miRNA expression profile and mRNA expression profile of CTS induced PDLCs were established.Bioinformatic analysis found important miRNAs that may be involved in the osteogenic differentiation of PDLCs under CTS.3.The role of miR-195-5p in the tension stress induced PDLCs osteogenesisObjectives:miR-195-5p has been reported involving in mechanotransduciton and osteogenic differentiation.In miRNA expression profile of CTS induced PDLCs differentiation,miR-195-5p is one of down-regulated miRNAs.However,the expression pattern and function of miR-195-5p in orthodontic tension stess induced-osteogenic differentiation is still unclear.The aim of this study was to investigate the expression pattern of miR-195-5p under tension stress in vitro and in vivo,explore the effect and analyze the regulational mechanisms on CTS induced-osteogenic differentiation.Methods&Results:The expression of miR-195-5p was significantly down-regulated under tension stress.Inhibition of miR-195-5p expression up-regulated osteogenic-related genes and promoted the accumulation of mineral nodules.Overexpression of miR-195-5p significantly down-regulated the expression of these genes and inhibited the accumulation of mineral nodules.WNT3A,FGF2 and BMPR1A were potential downstreams of miR-195-5p and were significantly up-regulated under CTS.Further analysis by DLA and Western blot indicated that miR-195-5p specifically bound to the 3’UTRs of WNT3A,FGF2 and BMPR1A and inhibited expression.Overexpression of WNT3A and BMPR1A can save the effect of miR-195-5p on osteogenic differentiation.Conclusions:Tension stress decreased miR-195-5p expression in PDLCs,resulted in the release of WNT3A and BMPR1A translational inhibition,and promoted the osteogenic differentiation.