Effects Of Silencing Cortactin Expression By Sirna On The Proliferation And Invasion Of Laryngeal Carcinoma Hep-2 Cell Line

Background and Objective: The cortical actin-binding protein cortactin participates in several functions including cytoskeleton system, cellular signal transduction and cell adhesion, and there is increasing evidence that it regulates tumor invasion and metastasis. However, the role played by cortactin in laryngeal carcinoma has not been clearly delineated. The purpose of this experiment was to investigate the effects of silencing cortactin expression on the proliferation and invasion of human laryngeal carcinoma cell line Hep-2 in vitro.Methods: A plasmid (pSilencer3.1-H1neo-cortactin) of a siRNA targeting cortactin was constructed by gene recombination. The recombinant gene was identified by DNA sequencing. The recombinant plasmid was stably transfected into Hep-2 cell line using liposome. After screening with G418, the localization of cortactin in Hep-2 cells was verified by immunocytochemistry and the siRNA interference efficiency of cortactin was determined by Western blot . The proliferation was measured by MTT assay and plate colony formation. Flow cytomery was used to determine the change of cell cycle. Transwell test was used to detect the migration and invasion ability of Hep-2 cells. Empty plasmid-transfected Hep-2 cells and normal Hep-2 cells were used as control.Results: The result of DNA sequencing shows that eukaryotic expression vector of a siRNA targeting cortactin gene is successfully constructed. Immunocytochemistry analysis shows cortactin protein only localizes to the cytoplasm in Hep-2 cells. The result of western blot shows that pcortactin-siRNA/Hep-2 cells can low express cortactin protein stably. Compared with Hep-2 cells, the expression of cortactin protein in pcortactin-siRNA/Hep-2 cells is 11.22% (P<0.01), its proliferation is inhibited (P<0.05) and the cloning efficiency decreases to 21.47%(P<0.01). The proportion of cells in the S phase is markedly decreased. The migratory and invasive ability is significantly decreased(P<0.01). Conclusion: 1. Pcortactin-siRNA/Hep-2 cells that could low express cortactin protein stably were established.2. Down-regulation of cortactin could inhibit the proliferation and invasive ability of Hep-2 cells.