Pygopus 2 promotes kidney cancer OS-RC-2 cells proliferation and invasion in vitro and in vivo

Abstract

Objective: Human Pygopus 2 (Pygo2) was recently discovered to be a component of the Wnt signaling pathway required for β-catenin/Tcf-mediated transcription. But the role of Pygo2 in malignant cell proliferation and invasion has not yet been determined.

Methods: Lentivirus-mediated small interfering RNA (siRNA) and vector-based overexpression were used to study the function of Pygo2 in OS-RC-2 cells. The resulted cells were subject to Western blotting assay, MTT assay, colony formation and cell invasion assays. Furthermore, renal cell carcinoma (RCC) models were established in BALB/c nude mice inoculated with OS-RC-2 cells. Immunohistochemistry (IHC) staining of matrix metalloproteinase-7 (MMP-7), matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) was performed in tumor tissue.

Results: Pygo2 gene was successful knocked down and overexpressed in RCC OS-RC-2 cells by using an shRNA and overexpressing vector, respectively. Overexpression of Pygo2 effectively promoted cell proliferation, colony formation and invasion in vitro. Knockdown of Pygo2 obviously inhibited xenograft tumor growth in nude mice. In addition, overexpression of Pygo2 increased the levels of MMP-7, MMP-9 and VEGF in the xenograft tumors.

Conclusion: Pygo2 has a role in promoting cell proliferation, invasion and metastasis, and may regulate angiogenesis via the Wnt/β-catenin signaling pathway.

Keywords: Matrix metalloproteinase-7; Matrix metalloproteinase-9; Pygo2; Renal cell carcinoma; Vascular endothelial growth factor.

Figure 1

Knockdown and overexpression of Pygo2 by Pygo2shRNA and an overexpressing vector respectively. (A) OS-RC-2 cells were infected with the indicated lentivirus including Pygo2-targeting shRNA (Pygo2shRNA), control shRNA (Pygo2scrRNA), Pygo2-overexpression (Pygo2OE), and GFP control. Bright field and fluorescence cell images were taken 2 days following transduction (100× magnification). (B) Protein extracted from the cells transduced as in (A) was subject to Western blotting assay. Protein levels of Pygo2 were detected by using an anti-Pygo2 antibody, and β-actin was detected to serve as a control for protein loading.

Figure 2

Effect of Pygo2 on OS-RC-2 cells growth in vitro. (A) Growth rates of OS-RC-2 cells as determined by MTT assay. (B) The effects of the Pygo2shRNA and overexpression of Pygo2 on colony formation of OS-RC-2 cells: a, Pygo2scrRNA; b, Pygo2shRNA; c, GFP; and d, Pygo2OE. (C) Invasion assay using BD BioCoat Matrigel Invasion Chambers. The stained cells were invasive cells in the lower chamber (200× magnification). (D) The number of migrating cells in (C) was quantified by photography. A 6-fold increase in the number of migrating cells in Pygo2OE was noted compared to Pygo2 knockdown in OS-RC-2 cell lines (p < 0.001).

Figure 3

The effect of Pygo2 knockdown and overexpression on tumor growth. (A) Nude mice inoculated with Pygo2shRNA, Pygo2scrRNA, GFP and Pygo2OE cells were sacrificed 5 weeks after inoculation. Note one mouse inoculated with Pygo2shRNA did not form tumor. (B) Tumor growth curves. Average volume of tumors in each group is plotted at different time points (p < 0.05). (C) Lung metastatic tumor was observed by light microscopy (Hematoxylin and Eeosin (H&E) staining, 200× magnification). (a) Pygo2shRNA group showing the effect of Pygo2 suppression on OS-RC-2 cells exhibit no lung metastatic tumor; (b) Control group; (c) Pygo2OE group showing the overexpression effect of Pygo2 on OS-RC-2 cells exhibit lung metastatic tumor (as shown by the arrow); (d) GFP group.

Figure 4

Immunohistochemical staining for Pygo2, MMP-9, MMP-7 and VEGF in tumor tissue. (A) Representative images of immunohistochemical staining of Pygo2, MMP-9, MMP-7 and VEGF expression (400× magnification). (B) Quantification of MMP-9-expressing cells (% of positive cells). The staining index was decreased in Pygo2shRNA-treated tumor tissues compared with that in control groups (**p < 0.01). (C) Statistical summary of MMP-7 positive cells, Pygo2shRNA protein expression was at a much lower percentage, a significant dose-dependent reduction in tumor tissues compared with control tumor tissues (***p < 0.001). (D) The rate of VEGF expression in Pygo2OE group is higher than that in Pygo2shRNA group (***p < 0.001).