Chiang Chi-Hsiang, Hou Ming-Feng, Hung Wen-Chun
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
Biochim Biophys Acta. 2013 Apr;1830(4):3067-76. doi: 10.1016/j.bbagen.2013.01.009. Epub 2013 Jan 16.
MiR-182 is a member of the miR-183 cluster located at human chromosome 7q32 region and is up-regulated in human cancers. We study the regulation of miR-182 expression and its oncogenic role.
MiR-182 level was investigated by real-time reverse transcription-PCR. Chromatin immunoprecipitation assay was used to confirm promoter binding of transcription factors. The correlation between miR-182 and RECK was analyzed by Western blotting, real-time RT-PCR and 3(')-untranslated region reporter assay. Zymography, matrix metalloproteinase activity, invasion and colony formation were used to study the tumorigenic activity.
MiR-182 is over-expressed in human breast tumor tissues and cell lines. Inhibition or knockdown of β-catenin reduced miR-182 level in MDA-MB-231 cells. ChIP assay confirmed the binding of β-catenin on miR-182 promoter. Anti-miR-182 increased the MMP inhibitor RECK protein in MDA-MB-231 cells while pre-miR-182 reduced RECK protein but not mRNA in normal mammary epithelial H184B5F5/M10 cells. Restoration of RECK protein by anti-miR-182 attenuated MMP-9 activity, cell invasion and colony formation. Ectopic expression of miR-182 inhibited restoration of RECK protein by β-catenin inhibitor indicating miR-182 is important for β-catenin-induced down-regulation of RECK. An inverse association between miR-182 and RECK was demonstrated in breast tumor tissues.
We provide evidence that miR-182 is up-regulated by β-catenin signaling pathway in breast cancer and its up-regulation increases tumorigenicity and invasiveness by repressing RECK.
Our data demonstrate for the first time that miR-182 expression is controlled by β-catenin. In addition, we identify a new miR-182 target RECK which is important for miR-182-induced tumorigenesis.
MiR-182是位于人类染色体7q32区域的miR-183簇的成员，在人类癌症中上调。我们研究了miR-182表达的调控及其致癌作用。
通过实时逆转录PCR研究miR-182水平。采用染色质免疫沉淀试验来确认转录因子与启动子的结合。通过蛋白质免疫印迹法、实时RT-PCR和3(')-非翻译区报告基因试验分析miR-182与RECK之间的相关性。使用酶谱法、基质金属蛋白酶活性、侵袭和集落形成来研究致瘤活性。
MiR-182在人类乳腺肿瘤组织和细胞系中过表达。β-连环蛋白的抑制或敲低降低了MDA-MB-231细胞中miR-182的水平。染色质免疫沉淀试验证实了β-连环蛋白与miR-182启动子的结合。抗miR-182增加了MDA-MB-231细胞中MMP抑制剂RECK蛋白的表达，而pre-miR-182降低了正常乳腺上皮H184B5F5/M10细胞中RECK蛋白的表达，但不影响其mRNA表达。抗miR-182使RECK蛋白恢复，减弱了MMP-9活性、细胞侵袭和集落形成。miR-182的异位表达抑制了β-连环蛋白抑制剂对RECK蛋白的恢复，表明miR-182对β-连环蛋白诱导的RECK下调很重要。在乳腺肿瘤组织中，miR-182与RECK呈负相关。
我们提供的证据表明，miR-182在乳腺癌中通过β-连环蛋白信号通路上调，其上调通过抑制RECK增加了肿瘤发生能力和侵袭性。
我们的数据首次证明miR-182的表达受β-连环蛋白控制。此外，我们鉴定出一个新的miR-182靶标RECK，它对miR-182诱导的肿瘤发生很重要。
