Focal Adhesion Kinase Expression In Human Colorectal Cancer Cell Behavior

Colorectal carcinoma is a common digestive malignant tumor which is one of main killers to the human being.In the last two decades,the morbidity of this cancer is rising steadily in most countries.In China,the morbidity of colorectal cancer shows a significant tendency towards going up as the living level improved.Tumor progression has strong correlation with activation and overexpression of pro-oncogene,inactivation and mutation of tumor suppressor gene,etc.So the genes associated with tumor development could be used as useful targets through RNAi to improve the diagnosis and treatment method.As the development of cellular and molecular biology,the considerable progresses have been established on the gene therapy of colorectal carcinoma.New targets offer more methods for the exploration,diagnosis and treatment of colorectal cancer.As a useful gene silencing method,RNAi suppressed the expression of corresponding gene at posttranscriptional level.Many researches reported that RNAi has a strong effect on gene inhibition. And its inhibition and specificity is stronger than that of antisense oligonucleotide.Because of the application prospect of RNAi in the gene silencing,especially the potential on the gene therapy, cancer research and drug development,RNAi had been used as a key technique in the anti-tumor research.In this dissertation,recombinant plasmids that produced siRNAs targeting FAK were used to transfected into Caco-2 cells to study the effects on FAK expression levels,apoptotic morphological changes,proliferation and motility of Caco-2 cells on the background of international research development and our previous results.The results got as follows:1.Four different siRNA sequences targeting human FAK mRNA and one non-target sequence were designed.Then recombinant plasmids pU6H1-GFP-siFAK were constructed by the company.2.Optimization of transfection efficiency:The recombinant plasmid pU6H1-GFP-siFAK was introduced into Caco-2 cells with Lipofectamine 2000.The following conditions were optimized: cell density,DNA amount,ratio of liposome and DNA,the forming time of liposome-DNA complex, incubation time of cell with liposome complex,serum,and the times of cell passage.The highest transfection efficiency was achieved with the following optimized conditions:at passage 2-5,2×10~5 cells,4μg DNA,2.5:1(V:W) ratio for liposome and DNA,30 min for complex formation,and 6 h for the incubation.Serum had no effect on the efficiency under these conditions.3.Effects on FAK gene expression:The changes of FAK expression levels and time effects were examined by RT-PCR,Western blotting and immunocytochemistry.These results indicated that FAK mRNA and protein level was down-regulated in Caco-2 cells significantly.The maximal inhibition on mRNA level was achieved at 48 h and on protein level at 72 h post transfection with time dependent.The four siFAK groups could down-regulate the FAK mRNA and protein level in different degrees,among which siFAK3 and siFAK4 groups were more effective.By immunohistochemistry,the inhibitory effect of siFAK3 and siFAK4 group for FAK protein level at 48,72 h post transfection was significant.4.Effects on behavior studies of Caco-2 cells:The apoptotic morphological changes,proliferation, and motility were investigated by Hoechst 33258 staining,MTT and wound healing assay.After Caco-2 cells transfected with pU6H1-GFP-siFAK,it induced typical apoptotic morphological changes.And ability of proliferation and motility of Caco-2 cells were significantly decreased in a time-dependent manner.