The Study Of The Function Of The Interaction Of Annexin A2and Stat3in Human Breast Cancer

ObjectiveAnnexin a2(Anxa2) is a calcium-dependent protein, which was widely and highly expressed in various cancer tissues, and playing multiple roles in tumor angiogenesis, cell proliferation, apoptosis, migration, invasion and adhesion. Our group’s preliminary study found that the expression of Anxa2could mediate the proliferation and migration ability of breast cancer cells, and regulate the expression of cell cycle-related proteins such as C-myc, Cyclin D1and the invasion-related proteins of MMP-2and MMP-9, which are the downstream response factors of Signal transducer and activator of transcription3(Stat3).Our early study suggested there is interaction between Anxa2and Stat3. And both Anxa2and Stat3are phosphorylated on tyrosine. So, we inferred that may be the phosphorylated Anxa2on23tyrosine phosphorylate the Stat3on its705tyrosine, which was subsequently activated and transport in nucleus and mediate the expression of downstream response proteins. In this study, Tyr23of Anxa2was mutated to an alanine residue, creating a non-phosphorylatable mutant or an aspartic acid mimicking constitutive phosphorylation by point mutation. Using infection or transfection technique, the monoclonal strains, which are stability expressing the corresponding proteins, were screened. So that we could further explore the roles of the Tyr23phosphorylation for Anxa2in regulating proliferation, migration and invasion in breast cancer cells. And look for new clues for the study of the mechanism of the interaction between Anxa2and Stat3.Methods:1. Scratch test was used to detect the influence of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D on the ability of migration in MCF-7cells in vitro.2. Migration and invasion assay in vitro was used to detect the effects of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D on the ability of migration and invasion in SK-BR-3and MCF-7cells.3. The RT-PCR technology was performed to detect the expression of mRNA of Stat3in the intraductal carcinoma, invasive ductal carcinoma of breast cancer patients, and analyze the correlation with the expression of mRNA of Anxa2.4. Employ the Co-immunoprecipitation technology, the interaction between Anxa2and Stat3in the SK-BR-3cells was detected.5. Western blotting was used to detect the effects of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D on the expression of Cyclin D1, which is a cell cycle-related protein.6. Taking Western blotting technology, the influence of Anxa2-WT, Anxa2-Y23A, and Anxa2-Y23D on the distribution and activation of Stat3in the nucleus and plasma of breast cancer SK-BR-3cells was detected.Results:1. The lentivirus plasmid pCDH-EGFP-Anxa2WT, which is wild-type of Anxa2and its mutants pEGFP-N3-Anxa2Y23A and pEGFP-N3-Anxa2Y23D were obtained.2. The pCDH-N3-Anxa2WT, pEGFP-N3-Anxa2Y23A, and pEGFP-N3-Anxa2Y23D successfully infected or transfected with the human breast cancer SK-BR-3and MCF-7cells.3. Anxa2-WT and its mutants Anxa2-Y23A and Anxa2-Y23D were stability expressed in the SK-BR-3and MCF-7cells.4. Anxa2-WT was detected by immunofluorescence technology to be mainly located in the cell membranes and plasma of SK-BR-3and MCF-7cells.5. The Anxa2-WT and Anxa2-Y23D enhanced the ability of proliferation and colony formation in breast cancer cells.6. The Anxa2-WT and Anxa2-Y23D improved the migrant and invasive ability of breast cancer cells.7. The expression level of Stat3mRNA in breast cancer invasive ductal carcinoma was significantly higher than adjacent tissues (P<0.0001) and intraductal carcinoma (P<0.0001); And there is a correlation between the expression Stat3mRNA and Anxa2mRNA in invasive intraductal carcinoma.8. According to the co-immunoprecipitation technology, there is an interaction between Stat3and Anxa2in the SK-BR-3cell.9. Taking advantage of Western blotting, we did not find there was any affection of Anxa2-WT, Y23D to the expression of Stat3and P-STAT3in the total lysate of SK-BR-3cells, but the expression of cell cycle related protein Cyclin D1was found to be up-regulated.10. Y23D was discovered to be able to promote the phosphorylation of Stat3in the nucleus by the Western blotting analysis.Conclusion:1. The phosphorylation of Anxa2on23Tyr plays an important role for its promotion of proliferation, migration and invasion in breast cancer cells.2. There is an interaction between Anxa2and Stat3in the SK-BR-3cells, and the phosphorylation of Anxa2on23Tyr was able to promote the phosphorylation of Stat3in the nucleus.