High-throughput mRNA and miRNA profiling of epithelial-mesenchymal transition in MDCK cells

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. N