MUC1 siRNA SILENCING IMPACT IN GASTRIC CANCER
CELLS PHENOTYPE
N. Costa, P. Paulo, R. Barbosa, H. Tsutsumida, T. Caffrey,
M.A. Hollingsworth, F. Santos-Silva*
Eppley Cancer Institute, UNMC, Omaha, NE, USA
IPATIMUP, Rua Dr Roberto Frias s/n, 4200-465 Porto, Portugal
INTRODUCTION. MUC1 is a trans-membrane glycoprotein expressed on
epithelial surfaces that was been associated with protection, cell signaling, cell-
cell interactions and cell migration (1). Over-expression and abnormal
glycosylation of MUC1 has been reported in several cancer models, including
gastric cancer (2). We evaluated the relevance of MUC1 for gastric cancer
tumorigenicity using a siRNA silencing strategy.
METHOD. We used small interfering RNAs (siRNA) to knock down MUC1
expression in MKN45 gastric cancer cell line. MUC1 expression levels in
silenced clones were confirmed by immunofluorescence and western-blot using
HMFG1 MAb and by real-time PCR. MUC1 silenced clones were evaluated for
proliferation (MTT assay), apoptosis (TUNEL assay) and invasion (Matrigel
invasion assay). Signaling pathways were evaluated with KinetworksTM protein
kinase multiblot analysis.
RESULTS. Using the RNAi strategy we stably down–regulated MUC1
expression. The MUC1 silenced clones (C1, C2) had significantly increased rates
of growth (proliferation), apoptosis and invasion activity when compared with
scramble control (SC) (figure 1). Furthermore these clones also showed
alterations in phosphorylation of proteins involved in oncogenic signaling
pathways: Erk1, decreased phosphorylation of T202 and Y204; Erk2, increased
phosphorylation of T185 and Y187; and cell cycle CDK1/2, increased
phosphorylation of T14, Y15, and T160/T161.