The Expression Of Annexin Ⅱ In Breast Cancer And Effect On Proliferation And Invasion Of Breast Cancer Cells

Objective:1.The study on the Annexin II and s100A10 levels in breast cancer tissues, and breast cancer cell lines (MDA-MB-435s,MDA-MB-231,ZR-75-30,MCF-7), is to explain the relations between Annexin II, s100A10 levels and invasive capacity in breast cancer cells. 2. To explore the effect of Annexin II - siRNA on the expression of Annexin II, s100A10 and c-Myc of breast cancer cells.3. To elucidate the role of Annexin II in proliferation, cell cycle arrest and invasion in human breast cancer cells.Methods:1. The expressions of Annexin II and s100A10 in normal breast tissues, ductal carcinoma in stiu as well as invasive ductal carcinoma were investigaed using immunohistochemistry (IHC); Invasive ability of four human breast cancer cells (MDA-MB-435s,MDA-MB-231,ZR-75-30,MCF-7) was assessed by Millicell chamber assay, and paving matrigel on the upper chamber; The levels of Annexin II and s100A10 mRNA, protein in breast cancers were detected respectively by use of polymerase chain reaction(PCR) and western blot.2. Small interference RNA (siRNA) duplexes targeting Annexin II were synthetized using chemical methods in vitro; MDA-MB-435s cells which were showed to overexpress Annexin II were transiently transfected by lipofectamin 2000, and the transfection efficiency was observed by fluorescence microscopy; Harvesting siRNA interferring cells, the levels of Annexin II,s100A10 and c-Myc were detected by real-time quantitative PCR,western blot and immunofluorescence(IF).3. Cell proliferation,cell cycle distribution and invasive capacity with or without plasminogen were detected by 3-(4,5-dimethylthiazol-2-yl)-5-diphenyl tetrazolium bromide (MTT),flow cytometer (FCM) and Millicell chamber assay respectively.Results:1. The immunohistochemical results revealed undetectable expressions of Annexin II and s100A10 in normal ductal epithelial cells;By sharp contrast, Annexin II and s100A10 were strongly overexpressed in the cytoplasm and plasma membrane of ductal epithelial carcinoma cells (P < 0.01);It was demonstrated a good correlation between the Annexin II,s100A10 expressions and the invasive character of the breast cancer . RT-PCR,Western blot results showed that Annexin II and s100A10 were overexpressed transcriptionally and translationally in high invasive breast cancer cell lines (MDA-MB-231,MDA-MB-435s,ZR-75-30) compared with that of low invasive breast cancer cell lines (MCF-7) (P < 0.01); Moreover, the level of s100A10 appeared tightly couple to Annexin II in breast cancer tissues or breast cancer cells. Confocal microscope investigated that Annexin II and s100A10 colocaliezd to plasma membrane and plasma;and the intensity of fluorescence staining was parallel to the invasive capacity of the breast cancer cells; No discrepancy of cellular distribution between the breast cancer cell lines was investigated .2. It was showed that the levels of Annexin II mRNA, protein of MDA-MB-435s cells treated with Annexin II -siRNA were obviously reduced compared with negative control cells and blank control (P<0.05);To my interest, knockdown of Annexin II resulted in down-regulation of s100A10 (P<0.05) as well as c-Myc proteins (P<0.05), but had no effect on s100A10 mRNA level (P>0.05).3. Compared with the blank and negative control groups, proliferation of cells treated with Annexin II -siRNA was remarkably inhibited at 48 h～96h(P<0.05),however, the cells proliferation had no alteration at 24h (P>0.05);The cell cycle distribution showed cell populations in the G0/G1 were obviously increased (P<0.05), along with decreased number of cells in S phase (P<0.05);In addition, Annexin II -siRNA cells invasive ability was obviously decreased (P<0.05). Conclusions:1. Annexin II and s100A10 were overexpressed in human breast cancer tissue and high invasive breast cancer cells; It was demonstrated a good correlation between the Annexin II,s100A10 expressions and the invasive character of the breast cancer.2. After breast cancer cells MDA-MB-435s were treated with Annexin II -siRNA, the expression of Annexin II,s100A10 and c-Myc were decreased, along with the inhibition of cancer cells proliferation and invasion.3. Annexin II might be a key contributor to the progress of breast cancer.