Anxa2 Expression Are Related To The Movement To The Cells Of Caco2 Micro Morphology And Protein Expression Of Regulation Research

Objective Colorectal cancer is one of the common malignant tumors. The cancer metastasis is the major cause of patient death. At present, the cure for colorectal cancer mainly contains traditional surgery resection, chemotherapy and radiotherapy. However, these treatments usually result in serious poisonous side effect and high recurrence efficiency. With the development of molecular and cellular biology, gene therapy employing RNAi technology brings new hope for curing malignant tumors. RNAi is defined as a mechanism of specific gene-knockdown mediated by double stranded RNA posttranscriptionally. Growing evidence shows that overexpression of ANXA2in colorectal cancer cells is closely related to cancer invasion and metastasis. In this study, we employed RNAi technology to inhibit the ANXA2gene expression in human colorectal cancer cells (caco2). Then explored the effects of ANXA2expression on the cell mobility related ultrastructure and cytoskeleton system of caco2cells and investigated the possible mechanisms in order to provide the experimental evidences for the gene therapy of colorectal carcinoma through ANXA2gene knockdown.Methods1. The ANXA2specific recombinant plasmids were introduced into caco2cells with the help of S-TranG transfection reagent.2. The cellular shape and surface ultrastructure were observed using scanning electron microscope at different time point post transfection.3. The cellular internal ultrastructure were observed using transmission electron microscope at different time point post transfection.4. The expression and distribution of F-actin, β-tubulin and Lamin B were detected by direct fluorescence labeling method, indirect immunofluorescent technique and immunocytochemistry technique respectively and the density (mean) of Lamin B were analyzed at different time point post transfection.Results1. Tranfection conditions were optimized for caco2cellls and the efficiency was achieved more than80%under the following conditions:60%-70%cell confluence before transfection,4.5μg plasmid and10μl S-TranG per30mm dish,10-15min plasmid/S-TranG complexes formation time.2. Scanning electron microscopy results show that the cellular shape was altered, meanwhile, the microvilli and pseudopodia structure were damaged after ANXA2gene knockdown. Moreover, the extent of the changes increased in a time dependent manner.3. Transmission electron microscopy results show that the cellular membrane system was damaged, the number and distribution of mitochondria and endoplasmic reticulum were altered and chromatic agglutination after ANXA2gene knockdown. Moreover, the extent of the changes increased in a time dependent manner.4. After ANXA2gene knockdown the cytoskeleton system was reorganized:the bundled F-actin was thin and scarce; the distribution of β-tubulin was disordered; the expression and the density (mean) of Lamin B were decreased. Moreover, the extent of the changes increased in a time dependent manner.Conclusion Down-regulation of ANXA2gene expression by RNAi technology could induce the cell mobility related ultrastructure alteration, cytoskeleton system remodling and transform the malignant phenotype of caco2cells. So, ANXA2is hopeful to become a target gene for colorectal cancer gene therapy. This study provide the experimental evidences for the gene therapy of colorectal carcinoma through ANXA2gene knockdown.