Long Noncoding RNA Moderates microRNA Activity to Maintain Self-Renewal in Embryonic Stem Cells. Mus musculus

Of the thousands of long non-coding RNAs expressed in embryonic stem (ES) cells, few have known roles and fewer have been functionally implicated in the regulation of self-renewal and pluripotency or reprogramming somatic cells to the pluripotent state. In ES cells, Cyrano is a stably expressed long intergenic non-coding RNA with no previously assigned role. We demonstrate that Cyrano contributes to ES cell maintenance, as its depletion results in loss of hallmarks of self-renewal. Delineation of Cyrano's network through transcriptomics revealed widespread effects on signaling pathways and gene expression networks that contribute to ES cell maintenance. Cyrano shares unique sequence complementarity with the differentiation-associated microRNA, mir-7, and mir-7 overexpression reduces expression of a key self-renewal factor to a similar extent as Cyrano knockdown. This suggests that Cyrano functions to restrain the action of mir-7. Altogether, we provide a view into the multifaceted function of Cyrano in ES cell maintenance. Overall design: RNA-seq on mouse R1 embryonic stem (ES) cells with two biological replicates transfected with an shRNA knockdown of Cyrano and two biological replicates transfected with a non-targeting control vector.

在胚胎干细胞（embryonic stem cell，ES细胞）表达的数千种长链非编码RNA（long non-coding RNA）中，仅有少数的功能已被阐明，而在调控细胞自我更新与多能性，或是将体细胞重编程至多能状态的过程中发挥功能性作用的更是寥寥无几。在ES细胞中，Cyrano是一种稳定表达的长基因间非编码RNA（long intergenic non-coding RNA），此前尚未明确其功能。我们的研究证实，Cyrano对ES细胞的维持具有促进作用：敲低该RNA会导致细胞丧失自我更新的标志性特征。通过转录组学解析Cyrano的调控网络，我们发现其对维持ES细胞的信号通路与基因表达网络具有广泛影响。Cyrano与分化相关的微小RNA（microRNA，mir-7）具有独特的序列互补性，而过表达mir-7会达到与敲低Cyrano相似的效果，即降低关键自我更新因子的表达水平。这表明Cyrano的功能是抑制mir-7的作用。综上，我们阐明了Cyrano在ES细胞维持过程中的多维度功能。整体实验设计：对小鼠R1 ES细胞开展RNA测序（RNA-seq），其中两组生物学重复转染靶向Cyrano的短发夹RNA（short hairpin RNA，shRNA）以实现基因敲低，另外两组生物学重复转染非靶向对照载体。