The Regulation And Mechanism Of MiR-19 On Osteoclast Differentiation

Objective 1.To study the effect of miR-19 on the differentiation process of osteoclasts;2.To detect which signaling pathways involved in the process of osteoclast differentiation regulated by miR-19;3.To find miR-19 targets to clarify the specific mechanisms that regulate the corresponding pathways during osteoclast differentiation.Methods 1.Through the over-expression and inhibition of miR-19 and the induction of osteoclast differentiation in RAW264.7 cell lines and BMM cells to understand the effects of TRAP staining and related osteoclast differentiation markers on the osteoclast differentiation.Furthermore,the expression of miR-19 in osteoclast differentiation was examined to understand the effect of osteoclast differentiation on its expression,and osteo-assay were performed to provide the effect of miR-19 on osteoclast function.2.Western blot and EMSA assays were used to examine the effect of RANKL-mediated protein and transcription factors on miR-19 overexpression,and to use AKT and NF-κB pathway inhibitors to intervene the above process to observe the expression of each protein and transcription factor.Change to understand the role of each signal pathway in the process of osteoclast differentiation under the influence of miR-19;3.Detection of TRAF6 ubiquitination and the detection of its inhibitory enzyme CYLD by co-immunoprecipitation to study the mechanism of miR-19 regulation of various pathways.And the effect compare with BMM cells over-expression miR-19 and BMM cells transfection si-CYLD.A dual luciferase reporter system was validated miR-19’s target gene.Results 1.Overexpression and inhibition of miR-19 can promote and inhibit the differentiation and bone resorption of osteoclasts,respectively,and miR-19 expression under physiological conditions is regulated by osteoclast differentiation.2.While RANKL stimulation after overexpression of miR-19,NF-κB,MAPK,and AKT pathways were all elevated.After interfered with AKT pathway inhibitors,no significant changes in the trend has detected of other pathway indicators except AKT.NF-κB Pathway inhibitors have a slight inhibitory effect on the process of osteoclast differentiation promoted by miR-19 overexpression.3.RANKL stimulation after miR-19 overexpression,TRAF6 binding ubiquitin index was increased,CYLD protein was inhibited after miR-19 was overexpressed,and si-CYLD effect was consistent with miR-19 overexpression.The dual luciferase reporter system detects that miR-19 binds to CYLD in vitro and exerts an inhibitory effect.Conclusion It was found that miR-19 plays a positive regulatory role in the process of osteoclast differentiation.Its target gene includes CYLD,and by inhibiting the expression of this protein,it promotes ubiquitination of TRAF6 and promotes downstream NF-κB,MAPK and AKT signaling pathway.The activation of the pathways exert positive regulatory effects.