Zhan Beilei, Zhan Yan, Wang Wei, Zhan Yunzhong, Liu Bingsheng
Department of Orthopedics, Quzhou People's Hospital, Quzhou, Zhejiang 324000, P.R. China.
Department of Science and Education, Quzhou People's Hospital, Quzhou, Zhejiang 324000, P.R. China.
Exp Ther Med. 2018 Jan;15(1):513-519. doi: 10.3892/etm.2017.5403. Epub 2017 Oct 31.
The aim of the present study was to assess miR-625 and Fas expression in normal and degenerative cervical cartilage endplate (CEP) tissues. Following biof-informatics analysis, the Fas gene was predicted to be one of the targets of miR-625. Quantitative PCR (qPCR) and western blotting were used to detect miR-625 and Fas expression in normal and degenerative CEP. A luciferase reporter assay was used to identify whether miR-625 could directly target the 3' untranslated region (3'-UTR) of Fas. Lentiviral overexpression and/or inhibition vectors of miR-625 (pre-miR-625)/antigomiR-625 were constructed to determine whether overexpression or inhibition of miR-625 could affect Fas and B-cell lymphoma 2 (Bcl-2) expression in cartilaginous endplate cells (CECs) and tissues. qPCR analysis demonstrated that miR-625 expression in degenerative CEP was significantly lower than in normal CEP tissue, while the production of Fas in degenerated CEP was significantly higher. Results from western blotting also showed a significant increase in Fas expression in degenerative CEP. miR-625 can bind directly to the 3'-UTR of the Fas gene. However, this inhibition was attenuated by a target mutation in the miR-625-binding site of the 3'-UTR of Fas mRNA. In addition, following transfection of CECs with pre-miR-625 and antigomiR-625, expression of Fas significantly decreased and increased, respectively, and Bcl-2 expression was upregulated and downregulated, respectively. Upregulation of miR-625 can inhibit Fas expression and further affect Bcl-2 expression in CEP degeneration, suggesting that miR-625-mediated inhibition of the Fas gene is important in cervical degeneration.
本研究的目的是评估miR-625和Fas在正常及退变颈椎软骨终板（CEP）组织中的表达情况。经过生物信息学分析，Fas基因被预测为miR-625的靶标之一。采用定量聚合酶链反应（qPCR）和蛋白质免疫印迹法检测正常及退变CEP中miR-625和Fas的表达。使用荧光素酶报告基因检测法来确定miR-625是否能直接靶向Fas的3'非翻译区（3'-UTR）。构建miR-625（pre-miR-625）/抗miR-625的慢病毒过表达和/或抑制载体，以确定miR-625的过表达或抑制是否会影响软骨终板细胞（CEC）和组织中Fas及B细胞淋巴瘤2（Bcl-2）的表达。qPCR分析表明，退变CEP中miR-625的表达显著低于正常CEP组织，而退变CEP中Fas的表达则显著升高。蛋白质免疫印迹法的结果也显示退变CEP中Fas的表达显著增加。miR-625可直接与Fas基因的3'-UTR结合。然而，Fas mRNA的3'-UTR中miR-625结合位点的靶标突变减弱了这种抑制作用。此外，用pre-miR-625和抗miR-625转染CEC后，Fas的表达分别显著降低和升高，Bcl-2的表达则分别上调和下调。miR-625的上调可抑制Fas的表达，并进一步影响CEP退变中Bcl-2的表达，提示miR-625介导的Fas基因抑制在颈椎退变中具有重要作用。
