A novel dual-fluorescence strategy for functionally validating microRNA targets in 3' untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212.
A novel dual-fluorescence strategy for functionally validating microRNA targets in 3' untranslated regions: regulation of the inward rectifier potassium channel K(ir)2.1 by miR-212.
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DOI：
10.1042/bj20120578
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发表时间：
2012-11-15
期刊：
影响因子：
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通讯作者：
Collins A
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文献类型：
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作者：
Goldoni D;Yarham JM;McGahon MK;O'Connor A;Guduric-Fuchs J;Edgar K;McDonald DM;Simpson DA;Collins A
通过microRNAs进行基因打靶对健康和疾病都很重要。我们建立了一种识别microRNA靶标的功能分析方法，并将其应用于心脏和血管疾病中调节失调的K+通道Kir2.1[KCNJ2(钾内向整流通道，亚家族J，成员2)]。将3‘非翻译区插入哺乳动物表达载体中的mCherry Red荧光蛋白编码序列下游。将microRNA序列插入pSM30表达载体，获得增强型绿色荧光蛋白作为microRNA表达的指示剂。将mCherry-3‘非编码区基因与含有microRNA的pSM30基质粒共转染人胚胎肾细胞系HEK-293细胞。该分析的原理是，通过自动图像分析确定，microRNA对3‘UTR的功能性靶向导致红色/绿色荧光强度比下降。该方法被已知的Kir2.1表达下调因子miR-1所验证，并被用于研究miR-212靶向Kir2.1 3‘非编码区。与非靶向对照相比，表达miR-212的细胞的红/绿比更低，这种影响通过突变预测的靶点而减弱。MIR-212还降低了HeLa细胞的内向整流电流和Kir2.1蛋白。这种新的分析方法与传统的基于荧光素酶的分析方法相比具有几个优点，包括更大的样本量，对时间进程研究的适应性以及对高通量筛选的适应性。
Gene targeting by microRNAs is important in health and disease. We developed a functional assay for identifying microRNA targets and applied it to the K+ channel Kir2.1 [KCNJ2 (potassium inwardly-rectifying channel, subfamily J, member 2)] which is dysregulated in cardiac and vascular disorders. The 3′UTR (untranslated region) was inserted downstream of the mCherry red fluorescent protein coding sequence in a mammalian expression plasmid. MicroRNA sequences were inserted into the pSM30 expression vector which provides enhanced green fluorescent protein as an indicator of microRNA expression. HEK (human embryonic kidney)-293 cells were co-transfected with the mCherry-3′UTR plasmid and a pSM30-based plasmid with a microRNA insert. The principle of the assay is that functional targeting of the 3′UTR by the microRNA results in a decrease in the red/green fluorescence intensity ratio as determined by automated image analysis. The method was validated with miR-1, a known down-regulator of Kir2.1 expression, and was used to investigate the targeting of the Kir2.1 3′UTR by miR-212. The red/green ratio was lower in miR-212-expressing cells compared with the non-targeting controls, an effect that was attenuated by mutating the predicted target site. miR-212 also reduced inward rectifier current and Kir2.1 protein in HeLa cells. This novel assay has several advantages over traditional luciferase-based assays including larger sample size, amenability to time course studies and adaptability to high-throughput screening.