Liu Wei, Zhang Yukun, Feng Xintong, Li Shuai, Gao Yong, Wang Kun, Song Yu, Yang Shuhua, Tu Ji, Shao Zengwu, Yang Cao
Department of Orthopedics, Tongji Medical College, Huazhong University of Science and Technology, Union Hospital, Wuhan 430022, China.
Department of Orthopedics, Tongji Medical College, Huazhong University of Science and Technology, Union Hospital, Wuhan 430022, China
Exp Biol Med (Maywood). 2016 Nov;241(17):1924-1932. doi: 10.1177/1535370216657444. Epub 2016 Jul 5.
Accumulating evidence indicates that miRNAs, a class of small non-coding RNAs, are implicated in the pathogenesis of various diseases such as cancer and intervertebral disc degeneration. The aim of this study was to investigate the expression and the biological function of microRNA-34a in intervertebral disc degeneration. In this study, microRNA-34a expression was assessed in nucleus pulposus specimens and in IL-1β-stimulated nucleus pulposus cells by real-time polymerase chain reaction. microRNA-34a functions were investigated by using gain and loss of function experiments in nucleus pulposus cells and a dual luciferase reporter assay in 293T cells. microRNA-34a was dramatically up-regulated in degenerative nucleus pulposus tissues and in IL-1β-stimulated nucleus pulposus cells when compared with controls. Furthermore, growth differentiation factor 5 was identified as a target of microRNA-34a. Aberrant expression of microRNA-34a inhibited growth differentiation factor 5 expression by direct binding to its 3'-untranslated region. This inhibition was abolished by mutation of the microRNA-34a binding sites. In addition, microRNA-34a silencing reversed IL-1β-induced decrease in type II collagen and aggrecan expression in nucleus pulposus cells. This effect was substantially suppressed by growth differentiation factor 5 silencing. Our results suggested that microRNA-34a inhibition prevents IL-1β-induced extracellular matrix degradation in human nucleus pulposus by increasing growth differentiation factor 5 expression. microRNA-34a inhibition may be a novel molecular target for intervertebral disc degeneration treatment through the prevention of nucleus pulposus extracellular matrix degradation.
越来越多的证据表明,微小RNA(miRNA),一类小的非编码RNA,与包括癌症和椎间盘退变在内的各种疾病的发病机制有关。本研究的目的是探讨微小RNA-34a在椎间盘退变中的表达及其生物学功能。在本研究中,通过实时聚合酶链反应评估了髓核标本和白细胞介素-1β(IL-1β)刺激的髓核细胞中微小RNA-34a的表达。通过在髓核细胞中进行功能获得和功能缺失实验以及在293T细胞中进行双荧光素酶报告基因测定来研究微小RNA-34a的功能。与对照组相比,微小RNA-34a在退变的髓核组织和IL-1β刺激的髓核细胞中显著上调。此外,生长分化因子5被确定为微小RNA-34a的一个靶标。微小RNA-34a的异常表达通过直接结合其3'-非翻译区抑制生长分化因子5的表达。微小RNA-34a结合位点的突变消除了这种抑制作用。此外,微小RNA-34a沉默逆转了IL-1β诱导的髓核细胞中II型胶原蛋白和聚集蛋白聚糖表达的降低。生长分化因子5沉默显著抑制了这种作用。我们的结果表明,微小RNA-34a抑制通过增加生长分化因子5的表达来防止IL-1β诱导的人髓核细胞外基质降解。微小RNA-34a抑制可能是通过预防髓核细胞外基质降解来治疗椎间盘退变的一种新的分子靶点。