Roles Of Fascin In The Human Esophageal Squamous Cell Carcinoma Cells

Esophageal squamous cell carcinoma is one of the most common malignancies. Up to now, its carcinogenesis mechanism is unclear. Fascin, an actin-bundling protein, induces membrane protrusions and increased cell motility in various transformed cells. The expression of fascin in epithelial neoplasms has been described only recently, and the role of fascin in esophageal squamous cell carcinoma (ESCC) is still unclear. To investigate the roles fascin plays in ESCC, we have used the pSUPER RNAi system to stably suppress the expression of the fascin gene in EC109, an ESCC cell line in which fascin is highly expressed and analyzed its effect on the organization of actin cytoskeleton, proliferation, invasive behavior, and explored some involved mechanisms. Materials and methods1. Expression and distribution of fascin protein were detected by Western blotting and immunofluorescence microscopy in EC109 cells and HeLa cells (a putative fascin over-expression tumor cell line).2. Several siRNAs targeted fascin gene were synthesized and inserted into pSUPER.neo. Positive clones were identified and verified by using restrictive cleavage and sequenced.3. Reconstructed RNAi plasmid hybrids using FuGENE 6 transfection reagent. G418 was added into the culture medium after 24h. Stable G418-resistant clones were obtained and the expanded cells were then used for subsequent studies.4. G418-screened EC109 cells were used for analysis of the silencing effect in protein level and RNA level by Western blotting and semi-quantity RT-PCR respectively.5. Fascin-downregulated cells were adopted to analyzed fascin effect on the organization of actin cytoskeleton, proliferation, invasive behavior, cell adhesion and explored some involvedmechanisms.6. The protein level of β-catenin and c-erbB-2 was detected by Western biotting.