The Study For The Molecular Mechanisms Of Annexin A2in Migration And Invasion In Drug-resistant Breast Cancer Cell

Objectives:The metastatic ability of breast cancer cells with chemo-resistant properties is increased in comparison with that of parental wild-type cells. The expression of AnnexinA2(Annexin a2). a36-kDa calcium-dependent phospholipids binding protein. is increased in metastatic tumors and was found to be associated with the phenotype of drug resistance and metastasis. In our study we used recombinant DNA techniques to up-regulate the expression of Annexin a2. The aim of the study is to investigate the effects of Annexin a2up-regulation on the proliferation, migration and invasion of human breast caner and to explore furtherly the mechanism by which Annexin a2regulates the growth and metastasis of breast cancer.Methods:1. We used gene cloning technology to up-regulate the expression of Annexin a2in human breast cancer and the expression of Annexin a2was detected by Western blotting.2. Using cell growth curve to detect the effects of Annexin a2up-regulation on proliferation of human breast cancer MCF-7cells.3. Colony formation assay was used to observe the influence of Annexin a2on colony formation rate of MCF-7cells.4. Flow cytometry analysis was used to detect the change of cell cycle.5. Wound healing assay was used to examine the effect of Annexin a2expression on migration.6. Invasion assay was performed to detect the influence of Annexin a2on human breast cancer invasive ability in vitro.7. In vivo tumorigenicity studies were performed to detect the effect of Annexin a2knockdown on the percentage of tumorigenesis.8. Western blotting was used to examine the expression of C-myc, Cylin Dl to explore Annexin a2may activate ERK1/2signaling pathways during tumor invasion and metastasis.Results: 1. We successfully constructed the eukaryotic expression vector named pcDNA3.1-Annexin a2to transfect human breast cancer MCF-7cells and3monoclonal cell strains with high expression of Annexin a2were screened. RNAi technology was used to transfect human breast cancer MCF-7/ADR cells and3monoclonal cell strains with low expression of Annexin a2were screened.2. The proliferation ability of MCF-7was promoted by the up-regulation of Annexin a2.3. The colony formation rate of MCF-7cell was increased by the up-regulation of Annexin a2.4. The migration and invasion of MCF-7cells were promoted with the up-regulation of Annexin a2.5. Decreased Annexin a2expression suppressed proliferation of MCF-7/ADR cells in vivo.6. Western blot analysis revealed a markedly lower expression of C-myc and Cyclin Dl in transfectants of Annexin a2RNAi while increased C-myc and Cyclin Dl were detected in clones with upregulated Annexin a2expression.7. The phosphorylation of ERK protein was decreased in siAnnexin a2/MCF-7/ADR cells and increased in MCF-7/Annexin a2cells, but the level of total ERK was not markedly altered with the phosphorylation of p-ERK.Conclusions:1. Up-regulation of Annexin a2increased the proliferation of MCF-7cells. The percentage of G0/G1was decreased while which of G2/M+S was increased. Colony formation was significantly increased in the Annexin a2-overexpressing transfectants of MCF-7cells.2. Annexin a2may play a key role in migration and invasion of MCF-7cells, and it might also contribute to the invasion of drug-resistant breast tumor cells.3. Annexin a2may play an important role in regulating the proliferation of MCF-7cells and MCF-7/ADR cells by influencing the expression of C-myc. Cylin D1via the activation of Erk1/2signaling pathways.