Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin
Overexpression of the microRNA hsa-miR-200c leads to reduced expression of transcription factor 8 and increased expression of E-cadherin
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DOI：
10.1158/0008-5472.can-07-1058
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发表时间：
2007-09-01
期刊：
影响因子：
11.2
通讯作者：
Brock, Graham J.
中科院分区：
文献类型：
--
作者：
Hurteau, Gregory J.;Carlson, J. Andrew;Brock, Graham J.
microRNA类似于22个核苷酸的序列，被认为与多个mRNA相互作用，导致翻译抑制或降解。我们先前报道了几种microRNA在哺乳动物细胞系中具有可变表达，我们更详细地研究了其中一种，miR-200 c。使用生物信息学和定量逆转录-PCR的组合来鉴定潜在的靶点，并揭示锌指转录因子转录因子8（TCF 8;也称为ZEB 1，delta EF 1，Nil-2-alpha）与miR-200 c的表达水平成反比。使用抗microRNA寡核苷酸的敲除实验增加了TCF 8水平，但具有非特异性作用。因此，为了研究靶预测，我们在选择的细胞系中过表达miR-200 c。通常，与正常人支气管上皮细胞相比，miR-200 c在非小细胞肺癌A549细胞中的表达水平较低。miR-200 c在A549细胞中的稳定过表达导致TCF 8的丢失，其调控靶点E-cadherin的表达增加，以及细胞形态的改变。在MCF 7（雌激素受体阳性乳腺癌）细胞中，存在miR-200 c和E-cadherin的内源性表达，但不存在TCF 8。相反，MDA-MB-231（雌激素受体阴性）细胞缺乏可检测的miR-200 c和E-钙粘蛋白（后者据报道是由于启动子区域甲基化），但表达TCF 8。该细胞系中miR-200 c的异位表达也降低了TCF 8的水平，恢复了E-钙粘蛋白的表达，并改变了细胞形态。由于E-eadherin的下调是上皮向间充质转化的关键事件，因此miR-200 c表达的缺失可能在侵袭性表型的起始中起重要作用，并且同样地，miR-200 c过表达具有逆转的潜力。
MicroRNAs are similar to 22-nucleotide sequences thought to interact with multiple mRNAs resulting in either translational repression or degradation. We previously reported that several microRNAs had variable expression in mammalian cell lines, and we examined one, miR-200c, in more detail. A combination of bioinformatics and quantitative reverse transcription-PCR was used to identify potential targets and revealed that the zinc finger transcription factor transcription factor 8 (TCF8; also termed ZEB1, delta EF1, Nil-2-alpha) had inversely proportional expression levels to miR-200c. Knockout experiments using anti-microRNA oligonucleotides increased TCF8 levels but with nonspecific effects. Therefore, to investigate target predictions, we overexpressed miR-200c in select cells lines. Ordinarily, the expression level of miR-200c in non-small-cell lung cancer A549 cells is low in contrast to normal human bronchial epithelial cells. Stable overexpression of miR-200c in A549 cells results in a loss of TCF8, an increase in expression of its regulatory target, E-cadherin, and altered cell morphology. In MCF7 (estrogen receptor-positive breast cancer) cells, there is endogenous expression of miR-200c and E-cadherin but TCF8 is absent. Conversely, MDA-MB-231 (estrogen receptor-negative) cells lack detectable miR-200c and E-cadherin (the latter reportedly due to promoter region methylation) but express TCF8. The ectopic expression of miR-200c in this cell line also reduced levels of TCF8, restored E-cadherin expression, and altered cell morphology. Because the down-regulation of E-eadherin is a crucial event in epithelial-to-mesenchymal transition, loss of miR-200c expression could play a significant role in the initiation of an invasive phenotype, and, equally, miR-200c overexpression holds potential for its reversal.