Effect of miR-223-3p on biological behavior of tendon stem cells under hypoxic conditions

Duan Cheng; Cheng Jie

doi:10.12307/2026.728

Abstract：BACKGROUND:The hypoxic microenvironment is a key regulator of tendon injury repair,but the molecular mechanism mediated by microRNAs(miRNAs)is not well understood. OBJECTIVE:To investigate the regulatory role and mechanism of miR-223-3p on the biological behavior of tendon stem cells under hypoxic conditions. METHODS:(1)The rat third-generation tendon stem cells were divided into control group(O₂ concentration 21%)and hypoxia group(O₂ concentration 1%).After 48 hours of culture,CCK-8 assay was used to detect cell proliferation.Muse apoptosis detection kit was used to detect apoptosis rate.RT-qPCR and western blot assay were used to detect the protein and mRNA expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor.(2)miR-223-3p targets were predicted by bioinformatics.The targeting and regulation relationship between miR-223-3p and VHL was verified by double luciferase assay.(3)The third-generation rat tendon stem cells were divided into five groups:normoxic miR-223-3p mimic group,normoxic mimic negative control group,normoxic inhibitor negative control group,hypoxic inhibitor negative control group,and hypoxic miR-223-3p inhibitor group.After culture under normoxia or hypoxia for 48 hours,the expression of hypoxia-inducible factor-1α,vascular endothelial growth factor,and VHL protein and mRNA was detected by RT-qPCR and western blot assay.Cell viability was determined by CCK-8 assay,and the apoptosis rate was analyzed by Muse cell apoptosis detection kit.The cell migration and invasion were evaluated by scratch assay and Transwell chamber assay. RESULTS AND CONCLUSION:(1)Compared with the control group,the tendon stem cell viability in the hypoxic group was significantly decreased(P<0.001),and the apoptosis rate was significantly increased(P<0.01).Compared with the control group,the mRNA expressions of miR-223-3p,hypoxia-inducible factor-1α and vascular endothelial growth factor in tendon stem cells in the hypoxic group were significantly increased(P<0.01).The expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor proteins in tendon stem cells in the hypoxia group were significantly increased(P<0.01).(2)Bioinformatics predicted that VHL had potential binding sites with miR-223-3p.Dual-luciferase assay showed that miR-223-3p could bind to VHL targeting.(3)Functional experiments showed that knockdown of miR-223-3p could up-regulate the mRNA and protein expression of VHL in tendon stem cells,down-regulate the mRNA and protein expressions of hypoxia-inducible factor-1α and vascular endothelial growth factor(P<0.01),improve the viability of tendon stem cells,reduce the rate of apoptosis,and promote cell migration and invasion(P<0.01),while overexpression of miR-223-3p was the opposite and aggravated hypoxic damage,which may be related to the activation of hypoxia-inducible factor-1α/vascular endothelial growth factor/VHL signaling pathway.