Annexin A7 and Its Related Protein Suppressor of Death Domains Regulates Migration and Proliferation of Hca-P Cells

Abstract

Objective: This study was to investigate whether annexin A7 (AnnexinA7, ANXA7) and its co-related protein tumor cell death domain silencer [suppressor of death domains (SODD)] regulates the migratory phenotype of liver cancer cells.

Materials and methods: In this experimental study, expression of ANXA7 in Hca-P cells, PANXA7 downregulated cells and PANXA7 unrelated sequence cells was detected by real-time quantitative polymerase chain reaction (PCR) at mRNA level and western blotting at protein level. Transwell migration and invasion assays were performed to determine the migratory phenotype.

Results: After inhibition of ANXA7 expression, expression of SODD protein was also significantly decreased (P<0.05). Transwell cell transfer experiments showed that number of tumor cells that penetrated into the cell membrane was significantly reduced after ANXA7 silencing (P<0.05). Transwell cell invasion assay showed that number of tumor cells penetrating into Matrigel was significantly reduced after ANXA7 down-regulation (P<0.05). The CCK8 assay was measured at 0, 24 and 48 hours, and proliferation rate of PANXA7 lower weir cells was slower than that of Hca-P cells and PANXA7 non-related sequence cells (P<0.05).

Conclusion: SODD expression was decreased with the down-regulation of ANXA7. Down-regulating ANXA7 in Hca-P cells decreased proliferation, migration and invasion of tumor cells.

Keywords: ANXA7; Lymph Node Metastasis; Transfection.

Fig.1

Silencing ANXA7 gene by using shRNA. A. Hca-P cells were transfected with expressing vectors pGPU6/GFP/Neo-shRNA and pGPU6/GFP/NeoshRNA- ANXA7 (scale bar: 100 μm). B. Expression levels of ANXA7 and SODD mRNA in Hca-P cells, Hca-P cells transfected with pGPU6/GFP/Neo-shRNA or Hca-P cells transfected with pGPU6/GFP/Neo-shRNA-ANXA7.

Fig.2

Western blot analysis of ANXA7 and SODD proteins in the indicated groups. Hca-P cells (a), Hca-P cells transfected with pGPU6/ GFP/NeoshRNA (b), and Hca-P cells transfected with pGPU6/GFP/NeoshRNAANXA7 (c). SODD; Suppressor of death domains.

Fig.3

The down-regulation effect of ANXA7 on cell migration was detected by Transwell assay. A. Representative images of transwell migration assay in Hca-P cells (a), Hca-P cells transfected with pGPU6/GFP/Neo-shRNA (b), and Hca-P cells transfected with pGPU6/GFP/Neo-shRNA-ANXA7 (c) (scale bar: 100 μm). B. Quantification of migrating cells in the above groups.

Fig.4

The effect of silencing ANXA7 on cell invasion. A. Representative images of transwell invasion assay in Hca-P cells (a), Hca-P cells transfected with pGPU6/GFP/Neo-shRNA (b), and Hca-P cells transfected with pGPU6/GFP/Neo-shRNA-ANXA7 (c) (scale bar: 100 μm). B. Quantification of invading cells in the above groups.

Fig.5

Inhibition of ANXA7 impaires the cell proliferation of Hca-P cells. CCK8 proliferation assay in Hca-P cells (blue group), Hca-P cells transfected with pGPU6/GFP/Neo-shRNA (yellow group), Hca-P cells transfected with pGPU6/GFP/Neo-shRNA-ANXA7 (grey group). h; Hour.