Experimental Study On Focal Adhesion Kinase Gene Silencing Promoting Anoikis Of Oral Cancer Cell

Inadequate or inappropriate cell-substrate contact triggers a subset of apoptotic cell death, termed anoikis. Resistance to anoikis is a characteristic of malignant cells that is associated with increased tumorigenesis and metastasis.Focal adhesion kinase (FAK), a 125kDA nonreceptor tyrosine kinase, is an important regulator of cell survival, invasion, migration, and cell cycle regulation. Moreover FAK is functionally important in transducing intracellular messages that are associated with growth factor signaling and cell-extracellular matrix interactions. FAK is also implicated in resistance to anoikis. Although the mechanisms by which FAK influences anoikis remain to be characterized fully, phosphatidylinositol 3-kinase, p130cas, p53, and ERK interactions have been identified in FAK-mediated cell survival. Artificial expression of constitutively active FAK has been reported to suppress anoikis in a variety of cells and can confer malignant properties on them. Conversely, the disruption of FAK activity with the use of FAK related nonkinase, a truncated dominant negative FAK homologue, has recently been reported to inhibit adhesion-dependent signaling in cardiac myocytes.For these reasons, we tested the hypothesis that FAK gene silencing would promote anoikis, reverse acquired anoikis-resistance, and inhibit metastasis of oral squamous carcinoma cells.Par I The construction of the siRNA expression vector and the silencing influence on FAK gene of Tca8113 cell lineObjective To construct the siRNA expression vector of FAK gene and inhibit the expression of FAK gene in tongue cancer cell line Tca8113 by RNA interfering. Methods According to the encoding sequence of mRNA of FAK, two pieces of oligonucleotide sequences were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pGCSilencer?-U6/Neo siRNA expression vector. After being identified by restriction enzyme, the recombinant plasmids pSilencer -FAK were transfected into Tca8113 cells, and the treated cells were selected by G418. Immuocytochemistry and Western blotting were used to validate FAK gene silencing efficiency in Tca8113 cell line. Results restriction enzyme showed that the oligonucleotide fragments were correctly inserted into pGCSilencer?-U6/Neo vector, and FAK expression of the transfected cells was significantly down- regulated by pSilencer-FAK. Conclusion The siRNA expression vector of FAK was successfully constructed and FAK expression of Tca8113 cells could be inhibited.Part II Focal adhesion kinase gene silencing promotes anoikis in human Tongue cancer cellObjective To test whether FAK gene silencing can promote anoikis and reverse acquired anoikis resistance in human Tongue cancer cell Methods FAK expression was assessed by immuocytochemistry and Western blot analysis. Anoikis was induced in Tca8113 (tougue squamocarcinoma cell) with the use of polyHEMA culture. FAK expression was suppressed by RNA interference. Anoikis was detected by flow cytometry. Results The cell line that were tested showed marked anoikis resistance, greater resistance being associated with higher levels of FAK expression. FAK gene silencing promoted anoikis remarkably in Tca8113 (P<0.01) . Conclusion FAK gene silencing suppresses anoikis resistance in Tongue cancer cell. FAK represents a potential target for novel antimetastatic therapies.