Rap2B promotes migration and invasion of human suprarenal epithelioma

Abstract

The aim of our study was to elucidate the role of Rap2B in the development of human suprarenal epithelioma and to investigate the effect of Rap2B on suprarenal epithelioma cells migration and invasion. We use tissue microarray and immunohistochemistry to evaluate Rap2B staining in 75 suprarenal epithelioma tissues and 75 tumor-adjacent normal renal tissues. And the expression of Rap2B protein in human suprarenal epithelioma cells and tissues was detected by western blot simultaneously. The role of Rap2B in suprarenal epithelioma cells migration and invasion was detected by using wound healing assay, cell migration assay, and matrigel invasion assay. After that, we performed western blot analysis and gelatin zymography to detect MMP-2 protein expression and enzyme activity. Our research showed that Rap2B expression was increased in tumor tissues compared with tumor-adjacent normal renal tissues. But no correlation was found between Rap2B expression and clinicopathological parameters. In addition, we found that Rap2B promoted the cell migration and invasion abilities, and Rap2B increased MMP-2 expression and enzyme activity in suprarenal epithelioma cells. Our data indicated that Rap2B expression is significantly increased in human suprarenal epithelioma and Rap2B can promote the cell migration and invasion abilities, which may provide a new target for the treatment of suprarenal epithelioma.

Fig. 1

Rap2B protein expression in suprarenal epithelioma cells and tissues. a In contrast to normal renal cells, the Rap2B expression is increased in 786-O cell lines. b Whole-cell protein extracts were further prepared from four paired tumor-adjacent normal renal tissues (N) and RCC tissues (T). The Rap2B protein level was determined by Western blot analysis

Fig. 2

Correlation between Rap2B expression and suprarenal epithelioma progression is shown. a Significant difference in Rap2B staining was observed between malignant tumor (MT) and tumor-adjacent normal renal tissue (AT). b The distribution of the difference in Rap2B staining (P<0.05, X² test). Magnification ×400

Fig. 3

Overexpression of Rap2B promotes the cell motility in suprarenal epithelioma cells. a Twenty-four hours after transfection, the expression of Rap2B was evaluated by Western blot in 786O cell and quantitative analysis shows that Rap2B protein was over-expressed after transfection. Actin was used as an internal control. b Wound-healing assay was executed that over-expression Rap2B promoted proliferation ability in wound closure compared with pcDNA3-control transfected group; c, d Cell migration assay and Matrigel cell invasion assay were performed after Rap2B over-expression in 786O cells. Rap2B over-expression promoted the ability to migrate and invade through Boy-den chamber. All experiments were carried out in triplicate. Data are shown as mean±SE. (EB, endogenous band) *P<0.05, **P<0.01, ***P<0.001

Fig. 4

Knockdown Rap2B inhibits the suprarenal epithelioma cells motility. a Forty-eight hours after transfection, the expression of Rap2B was evaluated by western blot in 786O cell and quantitative analysis shows that Rap2B protein was decreased after Rap2B knockdown. Actin was used as an internal control. b Wound-healing assay was executed after Rap2B knockdown in 786O cells. There was a significant delay in wound closure compared with pDNA3-control transfected group. c, d Cell migration assay and matrigel cell invasion assay were performed after Rap2B knockdown in 786O cells. The knockdown of Rap2B decreased the ability to migrate and invade through Boy-den chamber. All experiments were carried out in triplicate. Data are shown as mean±SE. *P<0.05, ***P<0.001

Fig. 5

Rap2B increases MMP-2 expression and ability in 786O cell. a Western blot analysis showed that MMP-2 expression was increased in suprarenal epithelioma cells after transfection of Rap2B. b In siRap2B and control group for 786O cell lines, the MMP-2 protein level was dramatically decreased in 786O-siRap2B cells. c, d Gelatin zymography confirmed that Rap2B over-expression can increase the MMP-2 ability compared to control. All experiments were carried out in triplicate. ***P<0.01