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Title: EpCAM knock-down in FaDu head and neck carcinoma cells      
Transcriptome or Gene expression
Epithelial cell adhesion molecule EpCAM is a single transmembrane protein, which is involved in numerous cellular processes including cell adhesion, proliferation, maintenance of stemness of embryonic cells and progenitors, migration, and invasion. Activation of signal transduction by EpCAM is warranted by regulated intramembrane proteolysis and nuclear translocation of the intracellular domain EpICD. Here, we describe matrix metalloproteinase 7 (MMP-7) as a target gene of EpCAM signalling via EpICD nuclear translocation. EpCAM and MMP-7 expression pattern and levels positively correlated in vitro and in vivo, and were strongly elevated in primary carcinomas of the head and neck area. Hence, MMP-7 is a novel target of EpCAM signalling. FaDu hypopharynx carcinoma cells were transiently transfected with a control siRNA or an EpCAM-specific siRNA. After two days, expression of EpCAM was assessed upon flow cytometry with antibodies, which specifically bind within the EGF-repeat in the extracellular domain of EpCAM (Balzar, et al., 1999). Compared to control siRNA, EpCAM siRNA induced a mean 43% reduction of EpCAM expression at the cell surface. Reduction of EpCAM expression upon siRNA treatment was confirmed by RT-PCR and qPCR. EpCAM down-regulation resulted in diminished cell proliferation assessed upon cell counting. The observed 43% decrease in cell surface expression of EpCAM translated in cell numbers diminished by 35%. This is a confirmation of earlier findings on the reduced proliferation of breast and colon cancer cell lines following siRNA-mediated knock-down of EpCAM (Maetzel, et al., 2009, Munz, et al., 2004, Osta, et al., 2004). Equal amounts of total RNA (RIN score = 10) from control- and EpCAM-siRNA-treated FaDu cells were next subjected to a single, explorative cDNA microarray analysis with the aim to identify potential target genes of the signal transduction cascade of EpCAM. All genes, which were up- or down-regulated by at least two-fold (>2-fold; 716 genes up-regulated, 552 genes down-regulated), were then subjected to a Panther classification software analysis ( Special emphasis was put on the regulation of genes associated with signalling pathways. Angiogenesis, Alzheimer's disease-presenilin pathway, Wnt signalling pathway, inflammation and cadherin signalling pathways were most strongly affected by EpCAM knock-down. mRNA levels of selected genes differently regulated following partial knock-down of EpCAM included the co-regulated genes for secreted frizzled-related protein 5, protocadherin 10, dickkopf homolog 2, MMP24, SPARC, Myst3, ErbB4, MMP12, WISP2, MMP7, Nanos1, WNT9A, and the counter-regulated genes for MMP11, fibronectin 1, DACT3, WNT2B, WNT5A, APC2, Spondin 1, and cateninĪ±2. All genes except for protocadherin 10, Myst3, ErbB4, and WNT2B were validated in RT-PCR assays. Since several matrix metalloproteases were regulated after EpCAM knock-down and EpCAM is known to correlate with invasion, we chose this protein family for in-depth analysis. Matrix metalloprotease 7 (MMP-7) is a soluble factor, which, unlike other family members, is preferentially expressed in epithelial cells (Wilson, et al., 1995). Since EpCAM also displays highest expression in epithelia, we chose to proceed with the analysis of MMP7. SiRNA-mediated knock-down of EpCAM resulted in a comparable 50% down-regulation of MMP7 mRNA levels in RT-PCR and qPCR. Overall design: Comparison of FaDu cells expressing EpCam vs FaDu cells with downregulated EpCam
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