Targeted-regulating of miR-204-5p on SOX4 to alleviate degeneration and senescence of nucleus pulposus cells
Objective:To investigate the biological effect and mechanism of miR-204-5p on intervertebral disc degeneration.Methods:Firstly,the digoxigenin biotin probe of miR-204-5p was constructed,and the tissue sections of nucleus pulposus with different degrees of degeneration were chemically stained to verify the expression of miR-204-5p in histology.Secondly,IL-1β was used to induce the degeneration model of intervertebral disc nucleus pulposus cells through stimulating at 5 ng/mL concentration for 0 h,6 h,12 h,24 h and 48 h respectively,and then the expression of miR-204-5p and miR-204-3p were detected by qRT-PCR.Bioinformatics database was used to predict the potential binding sequence of miR-204-5p and SOX4,and construct a double fluorescein reporter gene plasmid to verify the binding site.miR-204-5p mimics were used to transfect primary nucleus pulposus cells and qRT-PCR and Western blotting were used to detect the changes in downstream RNA and protein levels,and β-galactosidase staining was used to detect the senescence level of nucleus pulposus cells.Results:In the degeneration model of intervertebral disc nucleus pulposus cells induced by IL-1β,the expression of miR-204-5p was significantly up-regulated within 12 hours of stimulation,but the expression was significantly down-regulated after 12 hours(P<0.05).The digoxigenin biotin probe staining showed that the expression of miR-204-5p in the degenerated nucleus pulposus tissue was significantly down-regulated compared with that of normal nucleus pulposus tissue(13.7%vs.91.7%).The fluorescent activity of SW1353 cells co-transfected with SOX4 3'UTR wild-type(WT)reporter plasmid and miR-204-5p mimics was significantly lower than that of the control group(P<0.05),but for the cells with SOX4 3'UTR sequence mutation(MUT),the difference was neutralized.After overexpressing miR-204-5p,the expression level of Aggrecan was significantly up-regulated at the RNA and protein levels in nucleus pulposus cells,while the levels of potential target genes SOX4 and MMP3 significantly decreased(P<0.01),and the staining degree of β-galactosidase significantly increased.Conclusions:miR-204-5p may alleviate the aging of nucleus pulposus cells and maintain extracellular matrix homeostasis by targeted-inhibiting the post-transcriptional level of SOX4,which is expected to be a new target for the treatment of intervertebral disc degeneration.
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