| Background and ObjectivesGastric cancer is one of the most common malignant tumors in digestive tract. It has approximately42%morbility worldwide and emerges as the second leading cause of death in China, which is a big threat to human’s heath. Traditional diagnosis methods of gastric cancer include x-ray with the barium meal, B-ultrasonic examination, fibre-optic gastroscopy, CT and so on. However, these methods’sensitivity is so low that smaller tumors are easy to loss, putting patients at risk of missing the optimal treatment. Traditional therapy of gastric cancer has given priority to surgery, chemotherapy, radiotherapy, thermotherapy, immunotherapy as well as Chinese medicinal therapy. Nevertheless, gastric cancer patients usually face difficulties in clinical treatment and receive unsatisfactory therapeutic effects and poor prognosis, due to the high proliferation and metastasis. At present, studying tumor-related gene function has become the key point for cancer diagnosis in bio-medicine field. So, molecular targeted therapy for gastric cancer has created a new stage for cancer diagnosis, therapy and prognosis. RNAi is defined as a mechanism of specific gene knockdown, which is widely applied to study the gene function and therapy for curing malignant tumors. ANXA2plays important roles in tumor cell proliferation, invasion, metastasis, adhesion and the malignancy development of cancers. Our previous study on "the effects of ANXA2on the behaviors of SGC-7901cells" shows that ANXA2is excessively expressed in gastric carcinoma cells as to promote the proliferation of SGC-7901cells. In this study, we designed the siRNA targeting the mRNA of ANXA2to study the effects of ANXA2gene silencing on microstructures of SGC-7901cells. Then the cell microstructures was detected using immunofluorescence assay, immunocytochemistry assay, Coomassie brilliant blue staining assay and scanning electron microscope. To reveal the significance of ANXA2expression in the tumor development, this study was carried out based on the effect of ANXA2on the cell microstructures alteration and cytoskeleton system remodeling. We can gain more insight into the effect of ANXA2on tumor biology behavior, which could provide a more effectual idea for prevention, cure and prognosis of tumor. To achieve diagnosis and therapy of gastric carcinoma, it is necessary to explore the function of tumor related genes in the malignancy development of gastric cancer.Methods1. The ANXA2specific recombinant plasmids were introduced into SGC-7901cells in the condition of calcium phosphate transfection.2. The effect of ANXA2on the cell surface ultrastructure was observed using SEM.3. The expression and distribution of F-actin,β-tubulin, Vimentin and LaminB were detected using immunofluorescence assay, immunocytochemical staining and Coomassie brilliant blue staining assay. Moreover, mean fluorescence intensity of F-actin, β-tubulin, Vimentin and LaminB in SGC-7901cells was analyzed.4. The effect of ANXA2expression inhibition on the permeability of SGC-7901cells was detected using EB staining.5. The colocalization of ANXA2and FAK in SGC-7901cells was detected using laser scanning confocal microscope.Results1. The conditions of calcium phosphate transfection were optimized and the transfection efficiency was achieved more than90%under the following conditions: more than60%cell confluence before transfection,6μg plasmid,170μl calcium phosphate complex,20min complex formation time and16h incubation time.2. The expression of ANXA2was inhibited with siRNA by calcium phosphate transfection method in SGC-7901cells. SEM results showed that the cell shape was altered, the microstructures, such as pseudopodia/filopodia and microvilli, were weak and poor in ANXA2silencing SGC-7901cells. Meanwhile, the extent of the changes increased in a time dependent manner.3. The expression of ANXA2was inhibited; the cellular shape was altered; the bundle of F-actin was weak; the expression of (3-tubulin, Vimentin and LaminB was decreased; the distribution of β-tubulin and Vimentin was disordered. The density of all the above protein molecules was decreased.4. EB staining results showed high red fluorescence intensity in the nucleus after ANXA2down-regulation.5. Laser scanning confocal microscope results showed the colocalization of ANXA2and FAK in the cytomembrane, cytoplasm and nucleus of SGC-7901cells. ConclusionDown-regulation of ANXA2gene expression in SGC-7901cells could alter the cell shape, cytoskeleton system remodeling and transform the malignant phenotype of SGC-7901cells. ANXA2is necessary to maintain the cytoskeleton system of cancers, especially the F-actin polymerization, which leads to the alteration of the cell shape structures that are closely correlated with cell motility. Consequently, ANXA2could be considered as a diagnostic and curative potential target to be deeply investigated in gastric cancer. |
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