Regulation Of Microstructures ANXA2 Related Liver Cancer Cell Movement

Backgrounds and Objectives:Liver cancer is a malignant tumor of the top six in the world, with a high incidence and high mortality of basic characteristics, low diagnosis rate of early liver cancer and late treatment caused other lesions and infection is the main reasons that leading to death of liver cancer patients. Currently, more effective means of treatment in liver cancer, including liver resection, liver transplantation and other auxiliary chemotherapy, but there are certain side effects of these methods. With the development of modern molecular biology, gene therapy as a treatment in liver cancer diagnosis, treatment and prognosis has unparalleled advantages in recent years, and the key point for cancer diagnosis, treatment is study tumor-related gene function. RNAi technologies emerge as a new approach for studying tumor gene function, but also provides a more efficient means for further studying the pathogenesis of tumors, as well as provides new ideas for gene therapy. ANXA2is cell malignant transformation related genes, their abnormal expression is closely associated with tumor cell metastasis, invasion, adhesion, and the occurrence, development, transfer of cancer, large amounts of data show that ANXA2is high expression in hepatocellular carcinoma cells, also can be used as a serum marker for the diagnosis of HCC. ANXA2expression is inhibited in SMMC-7721cells by RNAi technology, which observation ANXA2regulate motility-related protein expression and distribution, intracellular movement microstructure changes. Study ANXA2as a target gene possibility for cancer gene therapy, and accumulation of the latest experimental data and evidence for liver cancer gene therapy. Exploreing gene function in the process of malignant transformation of liver cells and early stage of liver cancer occurrence, development and malignant transformation mechanism which help in breaking bottleneck of liver cancer diagnosis and treatment.Methods:1.To optimize calcium phosphate transfection conditions, targeting ANXA2interference recombinant plasmids transfected into SMMC-7721cells by optimized calcium phosphate transfection methods.2. Scanning electron microscopy observation of the movement structures and surface morphology structure of cells.3. Transmission electron microscopy analysis of interior submicroscopic structure of cells.4. Immunofluorescence marked the different structure of cytoskeleton, laser scanning confocal microscopy analysis of F-actin, P-tubulin expression and distribution changes.5. Immunocytochemistry and apoptosis assays were used to detect LaminB expression and distribution, morphological changes of the nucleus.Results:1.To optimize the conditions of calcium phosphate transfection, determine optimal transfection conditions,60%to70%cell confluence, swapped transfection medium1h before transfection,8μg plasmid dosage per dish,20to25min plasmid complexes formation,10h incubation time.2. Scanning electron microscopy results showed, ANXA2expression was inhibited, decrease of microvilli structures in cells, structural damage pseudopodia and other phenomena.3. Transmission electron microscopy results showed, down regulation of ANXA2expression, organelle structure changes in morphology structure and distribution, chromatin condensation.4. Immunofluorescence staining showed, ANXA2expression was inhibited, F-actin expression decrease, damage to the actin cytoskeleton, cell contact inhibition recovery in some extent, β-tubulin distribution organized deterioration, while also have a certain degree of depolymerization phenomenon.5. Immunocytochemistry and apoptosis detection results, down regulation of ANXA2expression, LaminB expression level decrease and its mean optical density values decline, nucleus showed a certain degree of characteristics of apoptosis.Conclusion ANXA2expression has an important regulatory role in the process of cell structure, cell malignant phenotype changes and cytoskeleton remodeling in SMMC-7721cells. So, ANXA2future is expected to become the target gene of the liver cancer treatment, which to play an important role in the hepatocellular carcinoma gene therapy. The topic provide new ideas and methods for the HCC therapy, and provide early experimental information and evidence in liver cancer gene therapy.