微RNA-210抑制人退行性变髓核细胞自噬影响椎间盘退行性变

Micro RNA-210 affect intervertebral disc degeneration by inhibiting autophagy of human degenerative nucleus pulposus cells

目的探讨微RNA-210(miRNA-210)对退行性变髓核细胞自噬的影响及其参与椎间盘退行性变(IDD)进程的可能机制。方法收集24例IDD患者(IDD组)和6例腰椎骨折患者(对照组)椎间盘组织进行分离,培养髓核细胞。通过实时荧光定量聚合酶链反应(FQ-PCR)检测髓核细胞中miRNA-210的表达,通过单丹磺酰尸胺(MDC)染色和蛋白质印迹法检测细胞自噬水平。通过FQ-PCR或蛋白质印迹法检测过表达或抑制miRNA-210对细胞自噬和细胞外基质代谢的影响。通过生物信息学手段寻找miRNA-210与自噬相关的靶基因及miRNA-210的结合位点,并应用双荧光素酶报告实验验证。结果与对照组相比,IDD组髓核细胞中miRNA-210表达量增加,细胞自噬水平降低。过表达miRNA-210会抑制髓核细胞自噬,同时降低Ⅱ型胶原(ColⅡ)及蛋白多糖表达量,升高基质金属蛋白酶-3(MMP-3)和MMP-13表达量。自噬抑制剂3-MA减弱miRNA-210对ColⅡ、蛋白多糖、MMP-3和MMP-13的调节作用。miRNA-210与自噬相关蛋白7(ATG7)存在结合位点,过表达miRNA-210通过沉默ATG7抑制细胞自噬,激活MMP-3和MMP-13,促进细胞外基质(ColⅡ和蛋白多糖)降解。结论在发生退行性变的椎间盘组织中miRNA-210表达量增加。过表达的miRNA-210可能通过直接作用于靶基因ATG7抑制细胞自噬,促进细胞外基质降解,推动IDD进程。

Objective To investigate the effects of microRNA-210(miRNA-210) on autophagy in human degenerated nucleus pulposus cells and the possible mechanisms of miRNA-210 involving intervertebral disc degeneration(IDD). Methods Intervertebral disc tissues of 24 IDD patients(IDD group) and 6 lumbar fracture patients(control group) were collected,and nucleus pulposus cells were cultured. The expression of miRNA-210 was detected by fluorescent quantitative real-time polymerase chain reaction(FQ-PCR),and the level of autophagy was detected by MDC staining and Western blotting. The effects of overexpression or inhibition of miRNA-210 on autophagy and extracellular matrix metabolism were detected by FQ-PCR or Western blotting. Target scan was used to predict the target gene of autophagy and binding site of miRNA-210. Dualluciferase reporter assay was performed to confirm the prediction. Results Compared with the control group,the expression of miRNA-210 in nucleus pulposus cells increased and the level of autophagy decreased in the IDD group. Overexpression of miRNA-210 significantly reduced the autophagy,downregulated the expression of type Ⅱ collagen(Col Ⅱ) and aggrecan, whereas upregulated the expression of matrix metalloproteinase-3(MMP-3) and MMP-13. Autophagy inhibitor 3-MA attenuated the regulation of miRNA-210 on Col Ⅱ,aggrecan,MMP-3 and MMP-13. A binding site existed between miRNA-210 and the 3'UTR of autophagy related 7(ATG7). Overexpression of miRNA-210 inhibited autophagy by silencing ATG7,thus activated MMP-3 and MMP-13,and promoted degradation of extracellular matrix(Col Ⅱ and aggrecan). Conclusion In degenerative intervertebral disc tissues,the expression of miRNA-210 increases. Overexpression of miRNA-210 may promote the process of IDD by directly acting on target gene ATG7 to inhibit autophagy and promote the degradation of extracellular matrix.