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  

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

作　　者：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] 

机构地区：[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

出　　处：《中国组织工程研究》2016年第2期248-253,共6页Chinese Journal of Tissue Engineering Research

摘　　要：背景:mi RNAs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mi RNAs表达谱的差异,确定退变性腰椎侧凸特异性相关的mi RNAs,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miR NA芯片筛查,挑选表达有差异的microR NA。随后,采用RT-q PCR技术对其进行验证。对表达显著差异的mi RNA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mi RNA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-qP CR验证,与正常对照相比,mi R-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,mi R-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mi R-491-5p影响基质金属蛋白酶9的表达。结果表明,下调的mi R-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;mi R-491-5p可成为治疗退变性腰椎侧凸的一个新的治疗靶点。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 micro RNA expression profile in degenerative lumbar scoliosis is rarely reported and understood. OBJECTIVE： To compare the micro RNA expression profile in the normal intervertebral disc and degenerative lumbar scoliosis and to identify degenerative lumbar scoliosis-specific micro RNAs, 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 micro RNAs in the nucleus pulposus tissues by micro RNA microarray was performed in 10 samples from each group. Subsequently, differentially expressed micro RNAs were validated using real-time quantitative RCR. The level of differentially expressed micro RNAs in the degenerative nucleus pulposus tissues was investigated. Then, the functional analysis of micro RNAs 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 micro RNAs that were differentially expressed（17 upregulated and 5 downregulated） in degenerative lumbar scoliosis patients compared with the controls. Following real-time quantitative RCR confirmation, mi R-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 mi R-491-5p promoted type Ⅱ collagen expression in nucleus pulposus cells. Bioinformatics target prediction identified matrix metalloproteinase-9 as a putative target of mi R-491-5p. Furthermore, luciferase reporter assays demonstrated that mi R-491-5p directly tar

关 键 词：脊柱侧凸 微RNAS 椎间盘 组织工程 组织构建 骨组织工程 退变性腰椎侧凸 mi R-491-5p 基质金属蛋白酶9 发病机制 

分 类 号：R318[医药卫生—生物医学工程]