miR-140-5P参与调控颈椎软骨终板退变

Involvement of miR-140-5P in Cartilaginous Endplate Degeneration in Cervical Vertebrae

目的探讨表达谱芯片初步分析microRNA差异表达是否参与调控颈椎软骨终板退变。方法选择25例颈椎软骨终板退变、需要行手术治疗的患者为病例组,15例无椎间盘软骨终板退变患者为对照组。选取病例组2例和对照组2例标本进行microRNA芯片差异表达分析。然后采用miRCURYTMLNA 18.0表达谱芯片进行原位杂交,由Axon GenePix 4000Bmicroarray扫描仪采集芯片荧光值。芯片结果经病例组15例和对照组15例标本进行Real-time PCR和Western blot验证。结果病例组发现22个上调和19个下调microRNA。Real-time PCR验证:病例组miR-140-5P表达量低于对照组的3倍、miR-140-5P的靶基因Adamts-5和Dnpep的表达量分别高于对照组的3倍和3.5倍(均P<0.05)。miR-140-5P与Adamts-5和Dnpep的miRNA表达呈负相关(r=-0.84、-0.76,均P<0.05)。Western blot结果示:病例组软骨终板Adamts-5和Dnpep的蛋白表达分别为0.66±0.46和0.57±0.32,均高于对照组软骨终板的0.22±0.06和0.26±0.08(均P<0.05)。结论本研究首次报道了miR-140-5P参与调控颈椎软骨终板退变。进一步研究miR-140-5P不仅有利于揭示软骨终板退变的分子生物学机制,而且其日后也可能成为治疗椎间盘退变的有效靶工具。

Objective To investigate whether the differential miRNA expression is correlated with cartilaginous endplate degeneration in cervical vertebrae.Methods Twenty-five patients needing surgical treatment for cartilaginous endplate degeneration in cervical vertebrae （case group）and 1 5 normal subj ects（control group）were selected in this study.The differential miR-NA expression was analyzed using the microarray in 2 patients from case group and 2 subj ects from control group.In situ hybridization was performed using miRCURY LNA Array（version, 18.0）,and fluorescence arrays were analyzed on an Axon GenePix 4000B scanner.The results of microarray analysis were further verified by real-time PCR and Western blot in 1 5 patients and 1 5 normal subjects.Results The expressions of 22 miRNAs were up-regulated and expressions of 19 miRNAs were down-regulated in patients with cartilaginous endplate degeneration in cervical ver-tebrae.Real-time PCR showed that the expression of miR-140-5P in case group was 3 times lower than that in control group.Furthermore,the expressions of miR-140-5P target gene Adamts-5 and Dnpep in case group were 3 and 3.5 times higher than those in control group,respectively（P〈0.05）.The expression of miR-140-5P was negatively correlated with the expressions of Adamts-5 and Dnpep（r=-0.84 and r=-0.76,respectively;P〈0.05）.Western blot showed that the pro-tein expressions of Adamts-5 and Dnpep in case group（0.66±0.46 and 0.57±0.32,respectively） were higher than those in control group（0.22±0.06 and 0.26±0.08,respectively）（P〈0.05）. Conclusion We reported,for the first time,an involvement of miR-140-5P in cartilaginous end-plate degeneration in cervical vertebrae.Further study on miR-140-5P will contribute to the clari-fication of molecular biological mechanisms of cartilaginous endplate degeneration and will be an efficient tool for targeted treatment of intervertebral disc degeneration.