Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR
Potential mRNA degradation targets of hsa-miR-200c, identified using informatics and qRT-PCR
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DOI：
10.4161/cc.5.17.3133
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发表时间：
2006-09-01
期刊：
影响因子：
4.3
通讯作者：
Brock, Graham J.
中科院分区：
文献类型：
--
作者：
Hurteau, Gregory J.;Spivack, Simon D.;Brock, Graham J.
使用基于锚定寡核苷酸（dT）的RT-PCR方法，我们定量了6个细胞系中10种microRNA的内源性表达。这鉴定了具有可变表达的miRNA，miR-200 c，其范围从在MDA-MB-231和HT 1080中检测不到到在MCF 7中高度表达。可变的表达提供了一个模型系统，以研究内源性之间的相互作用的miRNA和它们的计算预测的目标。因为如果相互作用导致切割或降解，则这些细胞系中预测的mRNA靶标和miR-200 c的表达水平应具有反比关系。为了选择用于分析的靶标，我们使用了Affytron表达数据和计算预测程序。亲和数据表明与3500种候选mRNA相似，在MCF 7中不存在，但在MDA-MB-231或HT 1080中存在。这些靶点与600个计算预测的miR-200 c靶点交叉参考，鉴定出20种潜在的mRNA。通过qRT-PCR对这些靶标和另外10种mRNA（单独使用预测程序排序选择）进行的表达分析揭示了与miR-200 c呈反比关系的4种mRNA，BIN 1、TCF 8、RND 3和LHFP。其余的大多数似乎没有降解（可能是翻译靶点）或在检查的细胞系中检测不到。最后，使用抗miRNA 2 '-0-甲基寡核苷酸（AMO）抑制miR-200 c导致靶之一转录因子TCF 8的表达增加。这些结果表明，单个miRNA可以直接影响重要转录因子的mRNA水平，尽管是以细胞系特异性的方式。需要进一步研究以在体内证实这一点并确定任何翻译效应。
Using an anchored oligo(dT) based RT-PCR approach we quantified endogenous expression of ten microRNAs in six cell lines. This identified a miRNA, miR-200c, with variable expression, ranging from undetectable in MDA-MB-231 and HT1080 to highly expressed in MCF7. The variable expression provided a model system to investigate endogenous interactions between miRNAs and their computationally predicted targets. As the expression level of the predicted mRNA targets and miR-200c in these lines should have an inverse relationship if cleavage or degradation results from the interaction. To select targets for analysis we used Affymetrix expression data and computational prediction programs. Affymetrix data indicated similar to 3500 candidate mRNAs, absent in MCF7 and present in MDA-MB-231 or HT1080. These targets were cross-referenced against similar to 600 computationally predicted miR-200c targets, identifying twenty potential mRNAs. Expression analysis by qRT-PCR of these targets and an additional ten mRNAs (selected using the prediction program ranking alone) revealed four mRNAs, BIN1, TCF8, RND3 and LHFP with an inverse relationship to miR-200c. Of the remainder, the majority did not appear to be degraded (and may be translational targets) or were undetectable in the cell lines examined. Finally, inhibition of miR-200c using an anti-miRNA 2'-0-Methyl oligonucleotide (AMO) resulted in an increase in expression of one of the targets, the transcription factor TCF8. These results indicate that a single miRNA could directly affect the mRNA levels of an important transcription factor, albeit in a manner specific to cell lines. Further investigation is required to confirm this in vivo and determine any translational effects.