Genome-wide integration of microRNA and the transcriptome during human alveolar epithelial cell transdifferentiation identifies SGK1 as novel target of miR- 424/503 [RNA-Seq]

The alveolar epithelium is comprised of two cell types, alveolar type 1 (AT1) and type 2 (AT2) cells, the latter being capable of self-renewal and transdifferentiation into AT1 fate to establish alveolar function. Micro-RNAs (miRNAs) re critical regulators of developmental pathways, including branching morphogenesis. To determine if miRNAs also play a crucial role in establishment/maintenance of cellular identity within the alveolar epithelium, we performed genome-wide analysis of miRNAs alongside transcriptomic alterations during transdifferentiation of human AT2 (hAT2) into AT1-like cells in vitro . Our results indicated that miRNAs are tightly correlated to known gene expression pattern changes during AEC differentiation. We were able to define three phases of miRNA expression during the trans differentiation process, “early”, “late” and “consistently” changed, which further sub-classified as up- or downregulated. miRNAs with altered expression at all time points during AT1-like differentiation were the largest subgroup, suggesting the need for consistent regulation of signaling pathways to mediate transdifferentiation . Target prediction analysis and integration with previously published gene expression data identified glucocorticoid signaling as the top pathway regulated by miRNAs. Serum/glucocorticoid kinase 1 (SGK1) emerged as a central regulatory factor with temporally-correlated loss of expression and gain of hsa-miR-424 and hsa-miR-503 expression. Functional validation demonstrated specific targeting of these miRNAs to the 3'UTR of SGK1. Taken together, our data show the time-related contribution of miRNAs to the alveolar transdifferentiation process and suggest the need for inhibition of glucocorticoid pathway signaling to achieve the AT1-like cell phenotype in vitro. The miRNA-mRNA interactions identified herein may help to establish AT2 to AT1 cell differentiation in vivo. Overall design: Total RNA was isolated from primary alveolar epithelial cells taken from human remnant transplant lung and purified as described previously (Marconett et al., 2013).

肺泡上皮由两种细胞类型构成，即肺泡1型（AT1）细胞与肺泡2型（AT2）细胞，其中后者具备自我更新能力，并可转分化为AT1细胞命运以建立肺泡功能。微小RNA（miRNAs）是包括分支形态发生在内的发育通路的关键调控因子。为明确miRNAs是否同样在肺泡上皮细胞身份的建立与维持中发挥关键作用，我们在体外将人源AT2（hAT2）细胞诱导为类AT1细胞的转分化过程中，同步开展了miRNAs的全基因组分析与转录组变化检测。
研究结果显示，miRNAs与肺泡上皮细胞（AEC）分化过程中已知的基因表达模式变化紧密相关。我们明确了转分化过程中miRNA表达的三个阶段，即“早期”“晚期”与“持续”变化阶段，并可进一步细分为上调或下调亚型。在类AT1细胞分化的全部时间点均出现表达改变的miRNAs为最大亚群，这提示需要持续调控信号通路以介导转分化过程。
靶标预测分析结合已发表的基因表达数据，将糖皮质激素信号通路鉴定为miRNAs调控的首要通路。血清/糖皮质激素激酶1（SGK1）作为核心调控因子出现，其表达呈现时间相关性的下调，而hsa-miR-424与hsa-miR-503的表达则同步上调。功能验证实验证实，这两种miRNAs可特异性靶向SGK1的3'非翻译区（3'UTR）。
综上，我们的研究揭示了miRNAs在肺泡转分化过程中的时间依赖性贡献，并提示体外诱导类AT1细胞表型需要抑制糖皮质激素通路信号。本研究鉴定的miRNA-mRNA互作关系或有助于在体内实现AT2细胞向AT1细胞的分化。
整体实验设计：总RNA提取自人类移植剩余肺组织的原代肺泡上皮细胞，并按照此前报道的方法进行纯化（Marconett等，2013）。