Cai Jia-Jun, Qi Zeng-Xin, Chen Ling-Chao, Yao Yu, Gong Yan, Mao Ying
Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China.
Department of Geriatrics, Huashan Hospital, Fudan University, Shanghai 200040, P.R. China.
Oncol Rep. 2016 Jan;35(1):284-90. doi: 10.3892/or.2015.4355. Epub 2015 Oct 27.
miR-124 and Capn4 are aberrantly expressed in glioblastoma multiforme (GBM) tissues. In the present study, we investigated miR-124 and Capn4 expression in GBM tissue specimens. The role of miR-124 and Capn4 in the migration and invasion of glioma cells in vitro was also examined. miR-124 and Capn4 expression in 20 GBM and 6 control brain specimens was examined using RT-qPCR and immuno-blotting. Data from The Cancer Genome Atlas were retrieved. Candidate mRNA target sites of miR-124 were predicted using TargetScan/microRNA and binding was examined using dual luciferase reporter assays. The U87 and U251 cells were transfected with scrambled microRNA, miR-124 mimics and/or pLenti-Capn4 prior to wound‑healing and Transwell invasion assays. Proteins involved in the epithelial-mesenchymal transition were examined using immunoblotting. The results showed that miR-124 was significantly downregulated in GBM tissues. Immunoblotting showed a marked upregulation of Capn4 expression in GBM tissues. The Spearman's correlation analysis revealed a negative association between miR-124 expression and Capn4 protein levels. TargetScan/microRNA predicted the miR-124 binding site in the nucleotide 440-446 region within the Capn4 3'-UTR, which was confirmed by luciferase assays. Wound‑healing and Transwell invasion assays demonstrated that Capn4 downregulation or miR-124 mimics suppressed the migration and invasion of glioma cells. Capn4 downregulation or miR-124 mimics reduced the level of phospho-FAK and MMP2, vimentin and N-cadherin in U87 cells. In conclusion, miR-124 was found to suppress the migration and invasion of glioma cells in vitro via Capn4.
微小RNA-124（miR-124）和钙蛋白酶4（Capn4）在多形性胶质母细胞瘤（GBM）组织中表达异常。在本研究中，我们调查了GBM组织标本中miR-124和Capn4的表达情况。同时也检测了miR-124和Capn4在体外对胶质瘤细胞迁移和侵袭的作用。采用逆转录定量聚合酶链反应（RT-qPCR）和免疫印迹法检测了20例GBM标本和6例对照脑标本中miR-124和Capn4的表达。检索了癌症基因组图谱的数据。使用TargetScan/微小RNA预测miR-124的候选mRNA靶位点，并通过双荧光素酶报告基因检测法检测其结合情况。在进行伤口愈合和Transwell侵袭试验之前，用乱序微小RNA、miR-124模拟物和/或慢病毒载体-Capn4转染U87和U251细胞。采用免疫印迹法检测参与上皮-间质转化的蛋白质。结果显示，GBM组织中miR-124明显下调。免疫印迹显示GBM组织中Capn4表达显著上调。Spearman相关性分析显示miR-124表达与Capn4蛋白水平呈负相关。TargetScan/微小RNA预测了Capn4 3'-非翻译区（UTR）中核苷酸440 - 446区域的miR-124结合位点，荧光素酶检测证实了这一点。伤口愈合和Transwell侵袭试验表明，Capn4下调或miR-124模拟物可抑制胶质瘤细胞的迁移和侵袭。Capn4下调或miR-124模拟物降低了U87细胞中磷酸化粘着斑激酶（FAK）和基质金属蛋白酶2（MMP2）、波形蛋白和N-钙黏蛋白的水平。总之，发现miR-124在体外通过Capn4抑制胶质瘤细胞的迁移和侵袭。
