Annexin A2 silencing inhibits invasion, migration, and tumorigenic potential of hepatoma cells

Figure 1 Annexin A2 expression level in hepatoma cells and silencing efficiency of small hairpin RNA in MHCC97-H cells. A: Representative Western blotting images of hepatocellular carcinoma cell lines and the normal hepatic cell line LO2. ^bP < 0.01 vs LO2; B: Representative Western blotting images of annexin A2 (ANXA2) silencing upon transfection of small hairpin RNA (shRNA). ^bP < 0.01 vs MHCC97-H; C: Representative immunofluorescence images of ANXA2 cellular distribution (× 400). DAPI: 4,6-diamidino-2-phenylindole.

Figure 2 Effect of small hairpin RNA-mediated annexin A2 silencing on proliferation and cell cycling of MHCC97-H cells. A: Cellular proliferation assay. ^aP < 0.05 vs the A₄₅₀ of MHCC97-H cells at 72 h; B: Cell cycle assay. P < 0.05 for percentage of MHCC97-H/annexin A2 (ANXA2)-small hairpin RNA (shRNA) cells in S phase vs that of MHCC97-H cells.

Figure 3 Suppressive effect of small hairpin RNA -mediated annexin A2 silencing on the invasion and migration potential of MHCC97-H cells. A: Representative images of invasive cells (stained with crystal violet) from: the MHCC97-H group; the MHCC97-H/control-small hairpin RNA (shRNA) group; the MHCC97-H/annexin A2 (ANXA2)-small hairpin RNA (shRNA) group; B: Representative images of cell migration. ^aP < 0.05 vs MHCC97-H cells.

Figure 4 Inhibitive effect of small hairpin RNA-mediated annexin A2 silencing on xenograft tumour growth in vivo. A: Representative images of xenografted and control mice and resected tumours. The MHCC97-H (untransfected) group (1); the MHCC97-H/control-small hairpin RNA (shRNA) group (2); the MHCC97-H/annexin A2 (ANXA2)-shRNA group (3); the blank control group (4). Tumorigenic nude mice appeared obviously emaciated, especially the those in the MHCC97-H group and MHCC97-H/control-shRNA group; B: Average tumour weights. ^aP < 0.05 vs MHCC97-H group; C: Tumour growth rates. ^aP < 0.05 vs MHCC97-H group at post-injection day 21; D: Representative immunohistochemical analysis and hematoxylin and eosin staining results (× 400). The density of ANXA2 staining (brown) in the cytoplasm of MHCC97-H/ANXA2-shRNA cells was obviously lower than that for the MHCC97-H cells or the MHCC97-H/control-shRNA cells. ANXA2 was mainly localized in the membrane of the MHCC97-H/ANXA2-shRNA cells, and localized in both the membrane and cytoplasm of the MHCC97-H cells and the MHCC97-H/control-shRNA cells. The morphological characteristics of subcutaneous xenograft tumours derived from MHCC97-H/ANXA2-shRNA cells were not fundamentally different from the other tumours.