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. 

Key words: ">tendon stem cell, miR-223-3p, hypoxia-inducible factor-1α, vascular endothelial growth factor, VHL, hypoxic environment