Transcriptome and targetome analysis in miR-155 expressing cells using RNA-seq. Homo sapiens

Previous studies have demonstrated the utility of microarray expression analysis to identify potential microRNA targets. Nevertheless, technical limitations intrinsic to this platform constrain its ability to fully exploit the potential of assessing transcript level changes to explore microRNA targetomes. High throughput multiplexed Illumina based next generation sequencing (NGS) provides a digital read out of absolute transcript levels and imparts a higher level of accuracy and dynamic range than microarray platforms. We used Illumina NGS to analyze transcriptome changes induced by the human microRNA, miR-155. This analysis resulted in a significantly larger inferred targetome than similar studies carried out using micro-array platforms. A comparison with 3' UTR reporter data demonstrated general concordance between NGS and corresponding 3' UTR reporter results. Non-harmonious results were investigated more deeply using transcript structure information assembled from the NGS data. This analysis revealed that transcript structure plays a substantial role in mitigated targeting and in frank targeting failures. With its high level of accuracy, its broad dynamic range, its utility in assessing transcript structure, and its capacity to accurately interrogate global direct and indirect transcriptome changes, NGS is a useful tool for investigating the biology and mechanisms of action of microRNAs.

既往研究已证实，基因芯片表达分析（microarray expression analysis）可用于筛选潜在的微小RNA（microRNA）靶标。然而，该平台固有的技术局限性，限制了其通过转录本水平变化分析以全面探索微小RNA靶转录组的潜力。基于Illumina平台的高通量多重下一代测序（next generation sequencing, NGS）可实现绝对转录本水平的数字化读取，且相较基因芯片平台具备更高的准确度与动态范围。本研究采用Illumina NGS技术，分析人类微小RNA miR-155诱导的转录组变化。结果显示，本研究推断得到的靶转录组规模显著大于采用基因芯片平台开展的同类研究。与3'非翻译区（3' untranslated region, UTR）报告基因数据的对比表明，NGS结果与对应3' UTR报告基因结果整体一致性良好。针对不一致的实验结果，本研究利用从NGS数据中组装得到的转录本结构信息开展了深入剖析。该分析揭示，转录本结构在靶向作用减弱与完全靶向失败过程中均发挥了关键作用。鉴于NGS具备高准确度、宽动态范围，可用于转录本结构分析，且能够精准探究全局直接与间接转录组变化，其是研究微小RNA生物学特性与作用机制的高效工具。