High-throughput mRNA and miRNA profiling of epithelial-mesenchymal transition in MDCK cells.
High-throughput mRNA and miRNA profiling of epithelial-mesenchymal transition in MDCK cells.
复制标题
DOI：
10.1186/s12864-015-2036-9
复制
发表时间：
2015-11-16
期刊：
影响因子：
4.4
通讯作者：
Macho-Maschler S
中科院分区：
文献类型：
--
作者：
Shukla P;Vogl C;Wallner B;Rigler D;Müller M;Macho-Maschler S
上皮-间质转化（EMT）是胚胎发育的重要过程，尤其是在原肠胚形成和器官形成过程中。此外，EMT在病理状况中被广泛观察到，例如，纤维化、肿瘤进展和转移。Madin-Darby犬肾（MDCK）细胞被广泛用于EMT和上皮可塑性的研究。MDCK细胞显示上皮表型，而致癌Ras转化的MDCK（MDCK-Ras）细胞经历EMT并显示间充质表型。对MDCK和MDCK-Ras细胞进行RNA-Seq和miRNA-Seq分析。通过qRT-PCR验证数据。进行基因签名分析以识别途径和基因本体论术语。对于选定的miRNA，进行靶预测。使用RNA-Seq，检测到大约一半的狗已知基因的mRNA。在Ras诱导的EMT期间对这些进行了差异调节筛选。我们进一步进行了基因签名分析，发现了基因本体论（GO）术语和途径对上皮极性很重要，并与EMT有关。在已鉴定的通路中，TGFβ1在许多EMT相关通路和生物学过程中作为中心信号因子出现。通过miRNA-Seq，发现大约一半的已知犬miRNA在MDCK和MDCK-Ras细胞中表达。此外，在差异表达的miRNAs中，检测到已知是EMT的重要调节因子的miRNAs，并预测了新的候选者。在miRBase中将我们的读段与其他物种的读段进行比对后，发现了新的狗miRNAs。重要的是，我们可以鉴定出25种具有稳定发夹结构的全新miRNAs。这些新的miRNAs中有两个差异表达。我们通过RT-qPCR验证了具有最高读取计数的两种新型miRNA。对一种在间充质MDCK-Ras细胞中高度表达的特定新型miRNA的靶向预测表明，它靶向上皮细胞连接复合物的组分。将最上调的miRNA的靶预测和MDCK-Ras细胞中靶的验证与通路分析相结合，使我们能够鉴定两种新的通路，例如，JAK/STAT信号传导和胰腺癌通路。这些途径不能仅通过RNA-Seq数据的基因集富集分析来检测。利用MDCK细胞的mRNA和miRNA以及MDCK细胞中Ras诱导的EMT的深度测序数据，鉴定了差异调节的mRNA和miRNA。许多已鉴定的基因在已知参与EMT的途径内。MDCK细胞中新的差异上调基因是干扰素刺激基因和参与Slit和Netrin信号传导的基因。确定了尚未与这些进程联系起来的新途径。Ras诱导的EMT的一个中心途径是TGFβ信号传导，其导致许多靶基因（包括miRNA）的差异调节。通过miRNA-Seq，我们鉴定了参与上皮细胞生物学或EMT的miRNA。最后，我们描述了全新的miRNAs及其靶基因。本文的在线版本（doi：10.1186/s12864-015-2036-9）包含补充材料，可供授权用户使用。
Epithelial-mesenchymal transition (EMT) is an important process in embryonic development, especially during gastrulation and organ formation. Furthermore EMT is widely observed in pathological conditions, e.g., fibrosis, tumor progression and metastasis. Madin-Darby Canine Kidney (MDCK) cells are widely used for studies of EMT and epithelial plasticity. MDCK cells show an epithelial phenotype, while oncogenic Ras-transformed MDCK (MDCK-Ras) cells undergo EMT and show a mesenchymal phenotype. RNA-Seq and miRNA-Seq analyses were performed on MDCK and MDCK-Ras cells. Data were validated by qRT-PCR. Gene signature analyses were carried out to identify pathways and gene ontology terms. For selected miRNAs target prediction was performed. With RNA-Seq, mRNAs of approximately half of the genes known for dog were detected. These were screened for differential regulation during Ras-induced EMT. We went further and performed gene signature analyses and found Gene Ontology (GO) terms and pathways important for epithelial polarity and implicated in EMT. Among the identified pathways, TGFβ1 emerged as a central signaling factor in many EMT related pathways and biological processes. With miRNA-Seq, approximately half of the known canine miRNAs were found expressed in MDCK and MDCK-Ras cells. Furthermore, among differentially expressed miRNAs, miRNAs that are known to be important regulators of EMT were detected and new candidates were predicted. New dog miRNAs were discovered after aligning our reads to that of other species in miRBase. Importantly, we could identify 25 completely novel miRNAs with a stable hairpin structure. Two of these novel miRNAs were differentially expressed. We validated the two novel miRNAs with the highest read counts by RT-qPCR. Target prediction of a particular novel miRNA highly expressed in mesenchymal MDCK-Ras cells revealed that it targets components of epithelial cell junctional complexes. Combining target prediction for the most upregulated miRNAs and validation of the targets in MDCK-Ras cells with pathway analysis allowed us to identify two novel pathways, e.g., JAK/STAT signaling and pancreatic cancer pathways. These pathways could not be detected solely by gene set enrichment analyses of RNA-Seq data. With deep sequencing data of mRNAs and miRNAs of MDCK cells and of Ras-induced EMT in MDCK cells, differentially regulated mRNAs and miRNAs are identified. Many of the identified genes are within pathways known to be involved in EMT. Novel differentially upregulated genes in MDCK cells are interferon stimulated genes and genes involved in Slit and Netrin signaling. New pathways not yet linked to these processes were identified. A central pathway in Ras induced EMT is TGFβ signaling, which leads to differential regulation of many target genes, including miRNAs. With miRNA-Seq we identified miRNAs involved in either epithelial cell biology or EMT. Finally, we describe completely novel miRNAs and their target genes. The online version of this article (doi:10.1186/s12864-015-2036-9) contains supplementary material, which is available to authorized users.