Aims Production of functional cardiomyocytes from pluripotent stem cells requires tight control of the differentiation process. Long non-coding RNAs (lncRNAs) exert critical regulatory functions in cell specification during development. In this study, we designed an integrated approach to identify lncRNAs implicated in cardiogenesis in differentiating human embryonic stem cells (ESCs). Methods and results We identified CARMA (CARdiomyocyte Maturation-Associated lncRNA), a conserved lncRNA controlling cardiomyocyte differentiation and maturation in human ESCs. CARMA is located adjacent to MIR-1-1HG, the host gene for two cardiogenic miRNAs: MIR1-1 and MIR-133a2, and transcribed in an antisense orientation. The expression of CARMA and the miRNAs are negatively correlated, and CARMA knockdown increases MIR1-1 and MIR-133a2 expression. In addition, CARMA possesses MIR-133a2 binding sites, suggesting the lncRNA could be also a target of miRNA action. Upon CARMA down-regulation, MIR-133a2 target protein-coding genes are coordinately down-regulated. Among those, we found RBPJ, the gene encoding the effector of the NOTCH pathway. NOTCH has been shown to control a binary cell fate decision between the mesoderm and the neuroectoderm lineages, and NOTCH inhibition leads to enhanced cardiomyocyte differentiation at the expense of neuroectodermal derivatives. Interestingly, two lncRNAs, linc1230 and linc1335, which are known repressors of neuroectodermal specification, were found up-regulated upon Notch1 silencing in ESCs. Forced expression of either linc1230 or linc1335 improved ESC-derived cardiomyocyte production. These two lncRNAs were also found up-regulated following CARMA knockdown in ESCs. Conclusions Altogether, these data suggest the existence of a network, implicating three newly identified lncRNAs, the two myomirs MIR1-1 and MIR-133a2 and the NOTCH signalling pathway, for the coordinated regulation of cardiogenic differentiation in ESCs.

中文翻译：

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保守的长链非编码 RNA CARMA 调节心肌细胞分化

目的 从多能干细胞产生功能性心肌细胞需要严格控制分化过程。长链非编码 RNA (lncRNA) 在发育过程中在细胞规范中发挥关键的调节功能。在这项研究中，我们设计了一种综合方法来识别与分化人类胚胎干细胞 (ESC) 的心脏发生有关的 lncRNA。方法和结果 我们鉴定了 CARMA（心肌细胞成熟相关的 lncRNA），这是一种控制人类 ESC 中心肌细胞分化和成熟的保守 lncRNA。CARMA 与 MIR-1-1HG 相邻，MIR-1-1HG 是两种心源性 miRNA：MIR1-1 和 MIR-133a2 的宿主基因，并以反义方向转录。CARMA 的表达与 miRNA 呈负相关，CARMA 敲低增加了 MIR1-1 和 MIR-133a2 的表达。此外，CARMA 拥有 MIR-133a2 结合位点，表明 lncRNA 也可能是 miRNA 作用的目标。在 CARMA 下调后，MIR-133a2 靶蛋白编码基因被协调下调。其中，我们发现了编码NOTCH通路效应子的基因RBPJ。NOTCH 已被证明可以控制中胚层和神经外胚层谱系之间的二元细胞命运决定，并且 NOTCH 抑制会导致心肌细胞分化增强，但会牺牲神经外胚层衍生物。有趣的是，发现两种 lncRNA，linc1230 和 linc1335，它们是已知的神经外胚层规范抑制因子，在 ESC 中的 Notch1 沉默后被发现上调。linc1230 或linc1335 的强制表达改善了ESC 衍生的心肌细胞的产生。在 ESC 中敲低 CARMA 后，这两种 lncRNA 也被发现上调。