The Study For Functions Of Annexin A2 In Human Breast Cancer

ObjectiveAnnexin a2 is a calcium dependent and phospholipid-binding protein overexpressed in various kinds of tumors and is one of the proteins expressed significantly differently between cancer and normal tissue and cells. Signal transducer and activator of transcription 3 (STAT3) processes the difunctionality of signal transducing and activating of transcription overexpressed in a variety of cancers, regulating the expression of proteins relating to proliferation, invasion and metastasis such as c-Myc, cyclin Dl, MMP-2 and MMP-9 and so on. In our study we used RNAi technology to knock down the expression of Annexin a2. The aim of the study is to investgate the effects of Annexin a2 down-regulation on the proliferation, migration and invasion of human breast caner and the interaction of Annexin a2 and STAT3 to explore furtherly the mechanism by which Annexin a2 regulates the growth and metastasis of breast cancer.Methods1. We used RNA interference technology to konckdown the expression of Annexin a2 in huamn breast cancer and the expression of Annexin a2 was detected by Western blotting.2. Using MTT to detect the effects of Annexin a2 down-regulation on proliferaton of human breast cancer MDA-MB-231 cells.3. Colony formation assay was used to observe the influence of Annexin a2 on colony formation rate of MDA-MB-231 cells.4. Flow cytometry anaylisis was used to detect the change of cell cycle.5. Wound healing assay was used to examine the effect of Annexin a2 expression on migration.6. Invasion assay was performed to detect the influence of Annexin a2 on human breast cancer invasive ability.7. Immunoprecipation and immunofluresence technology were used to detect the proteins interacting with Annexin a2.8. Werstern blotting was used to examine the expression of c-Myc, cyclin Dl, MMP-2 and MMP-9 to explore the mechanism of Annexin a2 regulationg the proliferation, migration and invasion of human breast cancer.Results1. ShRNA was used to transfects human breast cancer MDA-MB-231 and 3 monoclonal cell strains with low expression of Annexin a2 were screened.2. The proliferation ablity of MDA-MB-231 was inhibited by the knockdown of Annexin a2.3. The colony formation rate of MDA-MB-231 was decreased by the knockdown of Annexin a2.4. The migration and invasion of MDA-MB-231 cells were decreased with the down-regulation of Annexin a2.5. The invasion of MDA-MB-231 cells were inhibited with the down-regulation of Annexin a2.6. STAT3 were examined to interact with Annexin a2. The expression of c-Myc and cyclin Dl were decreased obviously with the down-regulation of Annexin a2.7. The expression of MMP-2 and MMP-9 were decreased significantly in the 3 monoclonal strains.Conclusions1. Annexin a2 interacted and coloalized with STAT3 in cytoplasm of MDA-MB-231 cells.2. The down expression of Annexin a2 inhibited the proliferation and the colony formation rates of MDA-MB-231. The percentage of Gl was increased while which of G2/M+S was decreased. The low expression of Annexin a2 inhibited the expression of c-Myc and cyclinDl, and it was approved that Annexin a2 by interacting with STAT3 affected the expression of c-Myc and cyclinDl to influence the proliferation of MDA-MB-231.3. The low expression of Annexin a2 impaired the migration and invasion of MDA-MB-231. The down expression of Annexin a2 inhibitd the expression of MMP-2 and MMP-9, and it was approved that Annexin a2 by interacting with STAT3 affected the expression of MMP-2 and MMP-9 to influence the invasion of MDA-MB-231.