| BackgroundMechanical stress and strain is the primary impetus for bone modeling and remodeling.Appropriate mechanical stimulation can promote the development and reconstruction of osseous tissue,so that the homeostasis of skeletal system can be maintained during the process of bone fracture repair,orthodontic tooth movement and distraction osteogenesis,etc.Mesenchymal stem cells(MSCs)as multipotent cells that commonly used in tissue engineering and regenerative medicine can be capable of differentiating into adipocytic,chondrocytic and osteocytic lineages according to differently changing microenvironment.Many studies have shown that suitable mechanical force can promote osteogenesis differentiation of BMSCs.MicroRNAs(miRNAs)are the class of noncoding single-stranded RNA molecules composed of approximately 21-25nucleotides.Functioning at the posttranscriptional level,miRNAs negatively regulate gene expression via degradation or translational inhibition of their target mRNAs by perfectly or imperfectly pairing with 3’untranslated region(3’UTR)of mRNAs.In recent years,many Mechanosensitive miRNAs have been indicated to participate in several cellular processes,including bone formation and homeostasis.In our previous work,we have revealed compelling evidence that miR-503-5p,as the mechano-sensitive miRNA,inhibits mechanical stretch-induced osteogenic differentiation and bone formation in OTM tension sides in vitro and vivo respectively.However,the underlying regulatory mechanisms of miR-503-5p in stretch-mediated osteogenic differentiation and bone formation are still not clear.ObjectiveTo furtherly shed light on the roles miR-503-5p play in mechanical stimulation-triggered osteoblast differentiation,in this study,we attempt to pridict,sift and verify the potential target gene of miR-503-5p and make sense of its furtherly functional mechanism meanwhile.Methods(1)Isolation,culture and differentiation of rBMSCs(2)Through bioinformatics method,the potential target genes of miR-503-5p were predicted via TargetScan,miRWalk,etc.The preliminary analysis was taken from the intersection of all predicted results and then these genes were subjected to GO term analysis.Meanwhile KEGG pathway annotation was also performed for these genes using the DAVID gene annotation tool.Finally,we select one as our target gene for further research in combination with related literature.(3)To explore expression pattern of the potential target gene of miR-503-5p by means of Luciferase reporter gene assay,qRT-PCR and Western blot.The data were analyzed using SPSS 16.0.(4)siRNA transfected:Cells transfected at a proper density with constructed siRNA and negative control were loaded under the optimal stress force,and then we detected BMPR1A and the osteogenesis related factors(ALP、Runx2、BSP、CollgenI)expression changes in protein level.Results(1)The success of vitro isolation,cultivation and identification of rBMSCs.Flow Cytometry tests displayed that the cells express markers of rBMSCs is CD44/CD90(+)and CD34/45(-).After osteogenic induction for 28 days,calcium salt deposition nodules can be observed after Alizarin red staining while after adipogenesis induction for 28 days,lipid droplet formed after red O staining.(2)It is more probably that Hoxa2,BCL2,IGF2,ACVR2A,SMAD1 and BMPR1A ect are the potential target genes of miR-503-5p.After comprehensive analysis,BMPR1A was choosed for further research.(3)Compared with negative control group,the ratio of luciferase activity of the group subjected to optically cycle stress force was higher,and the mRNA and protein expression level of BMPR1A increased similarly.(4)The mRNA and protein expression level of BMPR1A in the miR-503-5p mimics group were lower than those in the negative control group,while those were higher in the miR-503-5p inhibitor group after transient transfection with miR-503-5p inhibitor.(5)The results of Luciferase reporter gene assay reveal that compared with the group of miR-503-5p mimics-NC co-transfected with pmiR-RB-ReportTM-BMPR1A-3’UTR,the relative luciferase activity of the group co-transfected with miR-503-5p mimics and pmiR-RB-ReportTM-BMPR1A-3’UTR showed a significant decrease.On the contrary,the relative luciferase activity of the group co-transfected with miR-503-5p inhibitor and pmiR-RB-ReportTM-BMPR1A-3’UTR showed a significant increase compared with the group of miR-503-5p inhibitor-NC co-transfected with pmiR-RB-ReportTM-BMPR1A-3’UTR.(6)The protein expression levels of BMPR1A、ALP、Runx2、BSP and CollgenI in the group transfected with siRNA-BMPR1A were lower than those in the control group after being stimulated by optically cycle stress force.Conclusion(1)BMPR1A is the target gene of miR-503-5p during subjected to Cyclical stress。(2)The Negative regulatory role of miR-503-5p in Stretch-Induced Osteogenic Differentiation of rBMSCs is implemented through its target gene BMPRIA.Cyclical stress could result in the down-regulation of miR-503-5p,which further up-regulated its target gene BMPR1A participating in the osteogenic differentiation of rBMSCs. |
