微RNA-204-5p靶向调控SOX4缓解髓核细胞退变和衰老

Targeted-regulating of miR-204-5p on SOX4 to alleviate degeneration and senescence of nucleus pulposus cells

目的:探讨微RNA-204-5p(miR-204-5p)对椎间盘退行性变的生物学效应和作用机制。方法:收集2022年6—12月因脊柱疾病进行手术治疗的患者术中切除的废弃椎间盘组织。首先构建miR-204-5p的地高辛生物素探针,对正常与退行性变椎间盘髓核组织切片进行染色,并观察miR-204-5p的表达;然后采用5 ng/mL白细胞介素1β(IL-1β)分别刺激正常椎间盘髓核细胞0、6、12、24、48 h,构建不同退变程度髓核细胞模型,随后采用实时荧光定量PCR(qRT-PCR)检测miR-204-5p与miR-204-3p的表达;进一步利用生物信息学数据库预测miR-204-5p与SOX4潜在结合序列,并构建双荧光素酶报告基因质粒验证该位点;使用miR-204-5p过表达模拟物(miR-204-5p mimics)和mimics control对原代退行性变椎间盘髓核细胞进行转染,并采用qRT-PCR和蛋白质印迹法分别检测细胞外基质(ECM)代谢标志物表达水平,采用β-半乳糖苷酶染色检测退行性变椎间盘髓核细胞衰老水平。结果:RNA-地高辛生物素探针染色显示,miR-204-5p在退行性变椎间盘髓核组织中表达较正常椎间盘髓核组织显著下调(91.7%vs.13.7%);在IL-1β诱导髓核细胞退行性变模型中,刺激12 h内miR-204-5p表达显著上调,而刺激12 h后其表达显著下调(P<0.05);对SW1353细胞系共转染含SOX43'非翻译区(3'UTR)野生型(WT)报告序列质粒与miR-204-5p mimics的报告萤光素酶活性显著低于对照组(P<0.05),但是在SOX43'UTR序列突变后,该差异被中和;过表达miR-204-5p后,退行性变椎间盘髓核细胞在RNA和蛋白质水平上ECM合成代谢相关葡萄聚糖(Aggrecan)表达显著上调,而潜在靶基因SOX4与基质金属蛋白酶3(MMP3)表达均显著下调(P均<0.01)。且β-半乳糖苷酶染色结果显示,过表达miR-204-5p后退行性变椎间盘髓核细胞衰老程度缓解。结论:miR-204-5p可能通过靶向抑制SOX4的转录后水平而缓解髓核细胞衰老,并维持ECM稳态,有望作为椎间盘退行性变治疗的新靶点。

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 SOX43'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 SOX43'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.