中国组织工程研究

中国组织工程研究 ›› 2016, Vol. 20 ›› Issue (2): 248-253.doi: 10.3969/j.issn.2095-4344.2016.02.017

• 组织构建与生物活性因子 tissue construction and bioactive factors • 上一篇    下一篇

MicroRNA-491-5p调控基质金属蛋白酶9参与退变性腰椎侧凸的发病机制

王 磊1，李天旺1，刘建强2，刘晓宗3，王照国4，田 艳1，张永兴1，王 伟1   

  1. 1解放军第252医院脊柱外科，河北省保定市 071000；2保定市骨科医院骨科，河北省保定市 071000；3保定市第一中心医院，河北省保定市   071000；4保定市阜平县医院骨科，河北省保定市 073200
  • 收稿日期:2015-10-15 出版日期:2016-01-08 发布日期:2016-01-08
  • 作者简介:王磊，男，1982年生，河北省保定市人，汉族，2006年大连大学医学院毕业，硕士，主治医师，主要从事脊柱退变的研究。

MicroRNA-491-5p is involved in the pathogenesis of degenerative lumbar scoliosis by targeting matrix metalloproteinase 9

Wang Lei1, Li Tian-wang1, Liu Jian-qiang2, Liu Xiao-zong3, Wang Zhao-guo4, Tian Yan1, Zhang Yong-xing1, Wang Wei1   

  1. 1Department of Spinal Surgery, the 252nd Hospital of PLA, Baoding 071000, Hebei Province, China; 2Department of Orthopedics, Baoding Orthopedics Hospital, Baoding 071000, Hebei Province, China; 3Baoding First Central Hospital, Baoding 071000, Hebei Province, China; 4Department of Orthopedics, Hospital of Fuping County, Baoding 073200, Hebei Province, China
  • Received:2015-10-15 Online:2016-01-08 Published:2016-01-08
  • About author:Wang Lei, Master, Attending physician, Department of Spinal Surgery, the 252nd Hospital of PLA, Baoding 071000, Hebei Province, China

PDF

490

被引次数

6

摘要：

文章快速阅读：

文题释义：

腰椎侧凸：指脊柱的一个或数个节段在冠状面上偏离身体中线向侧方弯曲，形成一个带有弧度的脊柱畸形，通常还伴有脊柱的旋转和矢状面上后突或前突的增加或减少，同时还有肋骨左右高低不等平、骨盆的旋转倾斜畸形和椎旁的韧带和肌肉的异常，它是一种症状或X射线体征，可由多种疾病引起。
基质金属蛋白酶：是一个大家族，因其需要Ca2+、Zn2+等金属离子作为辅助因子而得名。其家族成员具有相似的结构，一般由5个功能不同的结构域组成：①疏水信号肽序列。②前肽区，主要作用是保持酶原的稳定。当该区域被外源性酶切断后，基质金属蛋白酶酶原被激活。③催化活性区，有锌离子结合位点，对酶催化作用的发挥至关重要。④富含脯氨酸的铰链区。⑤羧基末端区，与酶的底物特异性有关。其中酶催化活性区和前肽区具有高度保守性。

 

背景：miRNAs广泛参与蛋白表达的调控，在机体的多种生理和病理过程中发挥重要的作用，但是目前对其在椎间盘退变中的表达谱及发挥的作用知之甚少。

目的：比较退变性腰椎侧凸患者与正常对照组椎间盘组织中miRNAs表达谱的差异，确定退变性腰椎侧凸特异性相关的miRNAs，并对其进行功能验证。
方法：对获取的退变性腰椎侧凸57例患者手术髓核组织和腰椎骨折42例患者正常髓核组织依次进行总RNA提取，其中，各取10例进行miRNA芯片筛查，挑选表达有差异的microRNA。随后，采用RT-qPCR技术对其进行验证。对表达显著差异的miRNA进行探究。进一步对其进行功能验证，确定其与Ⅱ型胶原表达的关系。运用Western与荧光素酶报告系统进一步确定靶基因。
结果与结论：与正常对照相比，22条miRNA表达存在显著差异(17条表达 上调和5条表达下调)。随后进行RT-qPCR验证，与正常对照相比，miR-491-5p在退变性腰椎侧凸患者椎间盘组织中，表达显著下调。此外，miR-491-5p表达水平与椎间盘退变评分密切相关。高表达miR-491-5p促进Ⅱ型胶原表达。生物信息学软件证实，基质金属蛋白酶9为miR-491-5p理论上的靶基因。荧光素酶报告系统证实miR-491-5p影响基质金属蛋白酶9的表达。结果表明，下调的miR-491-5p通过调控基质金属蛋白酶9，导致椎间盘组织中Ⅱ型胶原的丢失，进而引起椎间盘退变及退变性腰椎侧凸；miR-491-5p可成为治疗退变性腰椎侧凸的一个新的治疗靶点。 

关键词: 组织构建, 骨组织工程, 退变性腰椎侧凸, miR-491-5p, 基质金属蛋白酶9, 发病机制

Abstract:

BACKGROUND: MicroRNAs are widely involved in the regulation of protein expression, and play a critical role in many physiological and pathological processes in the body. But microRNA expression profile in degenerative lumbar scoliosis is rarely reported and understood.
OBJECTIVE: To compare the microRNA expression profile in the normal intervertebral disc and degenerative lumbar scoliosis and to identify degenerative lumbar scoliosis-specific microRNAs, followed by functional validation.
METHODS: Total RNA samples were extracted from the nucleus pulposus tissues of 57 patients with degenerative lumbar scoliosis as experimental groups and the normal nucleus pulposus tissues of 42 patients with lumbar fractures as control group. An initial screening of differentially expressed microRNAs in the nucleus pulposus tissues by microRNA microarray was performed in 10 samples from each group. Subsequently, differentially expressed microRNAs were validated using real-time quantitative RCR. The level of differentially expressed microRNAs in the degenerative nucleus pulposus tissues was investigated. Then, the functional analysis of microRNAs in regulating collagen II expression was carried out. Western blot and luciferase reporter assay were also used to detect target genes.
RESULTS AND CONCLUSION: We identified 22 microRNAs that were differentially expressed (17 upregulated and 5 downregulated) in degenerative lumbar scoliosis patients compared with the controls. Following real-time quantitative RCR confirmation, miR-491-5p was significantly down-regulated in degenerative nucleus pulposus tissues in comparison with the controls. Moreover, its level was closely correlated with the pathological grading of disc degeneration. Overexpression of miR-491-5p promoted type II collagen expression in nucleus pulposus cells. Bioinformatics target prediction identified matrix metalloproteinase-9 as a putative target of miR-491-5p. Furthermore, luciferase reporter assays demonstrated that miR-491-5p directly targeted matrix metalloproteinase-9 and affected its protein expression in nucleus pulposus cells. These results show that the downregulation of miR-491-5p induces type II collagen loss by directly targeting matrix metalloproteinase-9, thereby resulting in degeneration of the intervertebral disc and degenerative lumbar scoliosis. This study also underscores the potential of miR-491-5p as a novel therapeutic target in degenerative lumbar scoliosis.