MicroRNA-1 Regulates Chondrocyte Phenotype And Suppresses Ihh Expression

Objective:(1)To investigate the result of miR-1 overexpression or inhibition on chondrocyte Ihh in chondrocytes cultured in vitro,and to explore its role in the degeneration of articular cartilage;(2)Analysis changes in the level of Ihh protein and gene levels and Hedgehog signaling pathway after miR-1 overexpression or inhibition,to investigate the role of miR-1 on Hedgehog signaling pathway regulating.Methods:(1)Analysis the result of miR-1 on the proliferation and differentiation of chondrocytes:(1)Isolation and culture of murine primary chondrocytes,Cell transfection of miR-1 mimic/miR-1inhibitor and control was performed at 100 n M.After 24 hours of culture,miR-1 was analyzed by Real-time PCR.(2)EDU and CCK-8 are performed to detect proliferation of chondrocytes.(3)the effect of miR-1 on the proliferation and differentiation : Cell transfection of miR-1 mimic/miR-1inhibitor and control was performed at 100 n M.After 24 hours of culture,m RNA expression of Col II,Col X,Ihh,SOX-9 and MMP-13 were analyzed by Real-time PCR.(4)The ELISA was used to detect the content of Ihh,COL-?,COL-?,and MMP-13 in the supernatant after transfection for 48 hours.(5)Immunofluorescence is performed to detection of Col II and Ihh of chondrocytes.(2)The role of miR-1 on Hedgehog signaling pathway regulating:(1)Construct a Ihh3'-UTR-luciferase reporter vector,and observe the effect of miR-1 on Ihh 3'-UTR luciferase activity by dual luciferase reporter gene assay to identify whether miR-1 and Ihh have direct interactions.(2)we transfected chondrocytes with miR-1 inhibitor,miR-1 inhibitor + si Ihh cotransfected with their control group,After 24 hours of culture,the expression of smo,gli-1,gli-2,gli-3,Pthrp geen was detected by RT-PCR.After 48 hours of culture,the expression of smo and gli-1 protein was detected by Western blot.Results: ? miR-1 significantly promoted chondrocyte proliferation and increased the expressions of matrix synthesis associated molecules Col II and Aggrecan.Besides,miR-1 significantly down-regulated the expressions of chondrocyte differentiation markers Col X and MMP-13.Moreover,miR-1 dose-dependently inhibited endogenous Ihh expression,and a negative correlation between miR-1 and Ihh expressions was observed.? miR-1 had two binding sites within the 3' untranslated region(UTR)of Ihh genes from various species.The luciferase reporter assay indicated that miR-1 post-transcriptionally suppressed Ihh expression,which was dependent on the binding of miR-1 to one of the two putative binding sites of Ihh 3' UTR.Furthermore,through inhibiting Ihh expression,miR-1 decreased the expressions of molecules downstream of Ihh in the Hedgehog signaling pathway in mouse thorax chondrocytes.Conclusion: In summary,our data indicate that miR-1 is able to regulate cell proliferation,the expressions of matrix synthesis and chondrocyte differentiation associated molecules in mouse thorax chondrocytes.miR-1 post-transcriptionally suppresses Ihh expression through binding to the 3' UTR of Ihh gene,and inhibits the Hedgehog signaling pathway.