MicroRNA-503-3p Affects Osteogenic Differentiation Of Human Adipose-derived Stem Cells By Regulating Wnt7b Under Cyclic Strain

MicroRNAs(miRNAs)play critical roles in osteogenic differentiation of mesenchymal stem cells by inhibiting mRNAs translation under cyclic strain.miR-503-3p in human adipose-derived stem cells(hASCs)was downregulated during osteogenic differentiation in vivo under cyclic strain in our previous studies,while it might target the Wnt/?-catenin signaling pathway.In this study,we explored the role of the miR-503-3p in osteogenic differentiation of hASCs under cyclic strain.Results of immunophenotypes by flow cytometry and multi-lineage potential confirmed that the cultured cells were hASCs.Results of luciferase reporter assay showed that miR-503-3p directly targeted the 3'-untranslated region(3'-UTR)of Wnt7 b,to regulate the expression levels of Wnt7 b.Under cyclic strain,gain-function or loss-function of miR-503-3p studies by mimic and inhibitor revealed that decreasing expression of miR-503-3p significantly promoted osteogenic differentiation of hASCs,whereas increased expression of miR-503-3p inhibited osteogenic differentiation.Furthermore,miR-503-3p high expression reduced the activity of the Wnt/?-catenin signaling pathway,as indicated by lowering expression of Wnt7 b and then inhibited ?-catenin in miR-503-3p-treated hASCs.By contrast,miR-503-3p inhibition activated the Wnt/?-catenin signaling pathway.Collectively,our findings suggest that miR-503-3p is a negative factor in regulating Wnt/?-catenin signaling pathway by Wnt7 b,which inhibits the osteogenic differentiation of hASCs under cyclic strain.Objective:To investigate the effects of mir-503-3p on the osteogenic differentiation of hASCs by targeting Wnt7 b.The results of this study will provide theoretical and experimental basis for promoting the application of mechanical factors in tissue engineering bone tissue construction.Methods:Human adipose tissue donated voluntarily by patients was collected,hASCs was isolated and obtained,and 3-6 generations of cells were selected for the experiment after preliminary identification of stem cell surface markers by flow cytometry and stem cell multidirectional differentiation.Cells proliferation was detected by Cell Counting kit-8(CCK-8).Cell cycle changes and apoptosis were determined with flow cytometry.Adipogenic differentiation were induced by culturing hASCs in adipogenic medium and examined by Oil Red O stain as an indicator of intracellular lipid accumulation.Osteogenic differentiation was induced by culturing hASCs in osteogenic medium and examined by the use of Alizarin Red stain as an indicator of osteogenesis.Alkaline phosphatase(ALP)staining was performed after osteogenic induction for 5 d to test osteogenic differentiation.Chondrogenic differentiation was induced by culturing hASCs in chondrogenic medium and assessed by Alcian Blue stain.The transfected cells were cultured in osteogenic media under uniaxial cyclic tensile strain(5%,0.5 Hz,2 h/d)using a computer-driven strain device(Flexcell5000?;Flexcell,USA).Luciferase reporter genes were performed to verify whether Wnt7 b was the target gene mediated by miR-503-3p.The hASCs were transient transfected by Wnt7b-siRNA interference clips.The interference efficiency and the changes of runt-related transcription factor 2(RUNX2),ALP,and secreted protein acidic and rich in cysteine(SPARC),Wnt7 b and ?-catenin expression were examined before and after the interference by qPCR analysis and Western-blot analysis.Results:Immunophenotypes and multi-directional differentiation supported the identity and purity of the cells from adipose tissue.Wnt7 b was a target gene mediated by miR-503-3p at both transcriptional and translational levels.Cyclic strain loading increased osteogenic differentiation of hASCs.Overexpression of Wnt7 b increased osteogenic differentiation of hASCs and knockdown of Wnt7 b inhibited osteogenic differentiation of hASCs.Suppression of miR-503-3p enhanced hASCs osteogenic differentiation and promotion of miR-503-3p suppressed hASCs osteogenic differentiation.The results revealed that the inhabitation of osteogenic differentiation regulated by miR-503-3p mimic was compensated by the overexpressed Wnt7 b.The results revealed that the enhancement of osteogenic differentiation regulated by miR-503-3p inhibitor was compensated by the downregulated Wnt7 b.Conclusions:miR-503-3p knockdown inhibited osteogenic differentiation of hASCs by upregulating Wnt7 b under cyclic strain,which provides theoretical and experimental basis for promoting the application of mechanical factors in tissue engineering bone tissue construction.