This study was set to study the molecular mechanism underlying how miR-200 regulates EGF/EGFR signaling to involve in epithelial-mesenchymal transition (EMT) in anaplastic thyroid cancer (ATC) cells. Loss-of-function experiments of EGFR silencing by siRNA transfection was performed. Transfection of pre-miR-200s or anti-miR-200s was used to increase or decrease miR-200 transcripts. Real-time PCR, Western blot, immunohistochemistry, and transwell experiments were performed to determine the role of miR-200s in EMT and its role in EGF/EGFR-mediated EMT in vitro and in vivo. EGF/EGFR signaling activation increased the expression of mesenchymal marker vimentin in Nthy-ori 3-1 cells and decreased the expression of endothelial maker E-cadherin. EGF stimulation led to increased RhoA expression in Nthy-ori 3-1 cells. EGFR silencing resulted in decreased RhoA expression in SW1736 and ARO cells. EGF stimulation led to down-regulation of miR-200s and EMT. Restoration of miR-200 expression by pre-miR-200a/c transfection reversed the process, including increased E-cadherin and decreased vimentin. Down-regulation of miR-200 by anti-miR-200 effectively reduced miR-200. Matrigel invasion assay proved that restoration of miR-200 expression counteracted invasiveness. EGFR silencing decreased invasiveness in SW1736 cells, while down-regulation of miR-200s restored invasiveness. Xenograft tumors of SW1736 cells with cotransfection of anti-miR-200s and EGFR siRNA which kept the similar E-cadherin and vimentin expression with the untransfected controls. In ATC cells, miR-200s play a central role in EGF/EGFR-mediated invasiveness in vitro and EMT in vivo.
Keywords: Anaplastic thyroid cancer; EGF/EGF; Epithelial–mesenchymal transition; Molecular mechanism; miR-200.