Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140

MicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We present here an experimental approach to target identification where the cartilage-specific miR-140 was overexpressed and silenced in cells it is normally expressed in separate experiments. Expression of mRNAs was profiled in both experiments and the intersection of mRNAs repressed by miR-140 overexpression and derepressed by silencing of miR-140 was identified. The intersection contained only 49 genes, although both treatments affected the accumulation of hundreds of mRNAs. These 49 genes showed a very strong enrichment for the miR-140 seed sequence implying that the approach is efficient and specific. 21 of these 49 genes were predicted to be direct targets based on the presence of the seed sequence. Interestingly, none of these were predicted by the published target prediction methods we used. One of the potential target mRNAs, Cxcl12, was experimentally validated by Northern blot analysis and a luciferase reporter assay. Each condition was profiled in triplicate. For over-expression experiment cells were transfected with siRNA-140 (miR-140 mimick) or siRNA-96 (as a control). For suppresion experiment cells were transfected with LNA-antimicroRNA-140 (suppressing endogenous miR-140) or LNA-antimicroRNA-449 (as a control).

微RNA（microRNAs，miRNAs）是一类调控信使核糖核酸（mRNA）表达的短链非编码RNA分子。由于miRNAs与其靶标间的同源性相对较低，miRNAs的靶标识别在计算层面存在较大挑战。本研究提出一种用于miRNA靶标识别的实验策略：在正常表达软骨特异性miR-140的细胞中，分别开展miR-140过表达与沉默实验。对两组实验中的mRNA表达情况进行了表达谱分析，并鉴定得到同时满足以下条件的mRNA：既在miR-140过表达时被抑制，又在miR-140沉默时去抑制。尽管两种处理均影响了数百条mRNA的积累，但该基因交集仅包含49个基因。这49个基因对miR-140的种子序列展现出极强的富集性，表明该方法高效且具有特异性。基于种子序列的存在，这49个基因中有21个被预测为miR-140的直接靶标。有趣的是，我们所使用的已发表靶标预测方法均未预测到这些基因中的任何一个。其中一条潜在靶标mRNA——Cxcl12，通过Northern印迹分析与荧光素酶报告基因检测得到了实验验证。每个实验条件均设置了三次重复实验。在过表达实验中，细胞分别转染了siRNA-140（miR-140模拟物，miR-140 mimick）与作为对照的siRNA-96；在沉默实验中，细胞分别转染了靶向内源性miR-140的锁核酸（Locked Nucleic Acid，LNA）抗miRNA试剂（LNA-antimicroRNA-140）与作为对照的LNA-antimicroRNA-449。