Suppressive effects of RASAL2 on renal cell carcinoma via SOX2/ERK/p38 MAPK pathway

Abstract

Metastatic renal cell carcinoma (RCC) is associated with poor prognosis. Ras protein activator like 2 (RASAL2) protein has been previously demonstrated to serves as a tumor suppressor in a variety of malignancies. Therefore, the aim of the present study was to investigate the role of RASAL2 in RCC. Reverse transcription-quantitative PCR, western blot analysis and immunohistochemistry were performed to measure mRNA and protein expression in RCC tissues, whilst immunofluorescence and western blotting were performed to evaluate protein expression in RCC cells. A Cell Counting Kit-8 and 5-bromo-2'-deoxyuridine staining were applied to determine cell viability, and Transwell assays were conducted to measure RCC cell invasion and migration. RASAL2 expression was identified to be downregulated in RCC tissues, which associate negatively with RCC pathological grade. Sox2 expression, in addition to ERK1/2 and p38 MAPK phosphorylation, were demonstrated to be increased in RCC tissues. In RCC cells, RASAL2 overexpression decreased the expression of Sox2 and the activation of ERK1/2 and p38 MAPK. Physiologically, RASAL2 overexpression decreased RCC cell viability, invasion and migration. The expression of metalloproteinase-2/9 and tissue inhibitor of metalloproteinase 1 were also identified to be decreased and increased by RASAL2 overexpression, respectively. By contrast, RASAL2 knockdown exerted opposite effects on RCC cells compared with those observed following RASAL2 overexpression. RASAL2 expression decreased RCC cell viability, migration and invasion, which was demonstrated to be associated with the inactivation of SOX2/ERK1/2/p38 MAPK signaling. These results suggest that RASAL2 may potentially serve as a potential target for the development of novel therapeutic intervention strategies against RCC.

Keywords: extracellular signal-regulated kinase; invasion; migration; p38 mitogen-activated protein kinase; ras protein activator like 2; renal cell carcinoma.

Figure 1

RASAL2 expression was decreased in RCC tissues. (A) Comparison of RASAL2 mRNA expression in normal and RCC tissues, measured using reverse transcription-quantitative PCR. (B) Comparison of RASAL2 protein expression in normal tissue and RCC tissues, measured using western blotting. (C) Comparison of RASAL2 protein localization in normal and RCC tissues, as measured using immunohistochemical staining. Scale bars=100 µm (upper panel); Scale bars=50 µm (lower panel). ^*P<0.05 vs. normal RCC tissue. ^@P<0.05 vs. low-grade RCC. ^#P<0.05 vs. middle-grade RCC. RCC, renal cell carcinoma; RASAL1, ras protein activator like 2.

Figure 2

SOX2, p-ERK and p-p38 MAPK expressions were notably elevated in RCC tissues. Protein expression levels of SOX2, ERK1/2, p-ERK1/2, p38 and p-p3 were measured in RCC tissues by western blotting analysis. And the relative expressions were calculated in accordance with the gray value. ^*P<0.05 vs. normal RCC tissue. ^@P<0.05 vs. low-grade RCC. ^#P<0.05 vs. middle-grade RCC. RCC, renal cell carcinoma; p-, phosphorylated.

Figure 3

Overexpression and knockdown of RASAL2 expression in ACHN cells. (A) RASAL2 expression was evaluated in ACHN cells using reverse transcription-quantitative PCR, (B) western blotting and (C) immunofluorescence staining following transfection with plasmids expressing either the RASAL2 protein or shRASAL2. Magnification, x200. ^*P<0.05 vs. Control group. ^#P<0.05 vs. scramble group. Blank, un-transfected control; Control, control pcDNA.31 plasmid; sh, short hairpin RNA; scramble, scrambled shRNA; RASAL2, ras protein activator like 2; shRASAL2, RASAL2 shRNA.

Figure 4

Effect of manipulating RASAL2 expression on RCC cell viability, migration and invasion. (A) Following RASAL2 overexpression or knockdown in ACHN cells, cell viability was measured using Cell Counting Kit-8 assay. (B-D) ACHN cell migration and invasion ability was evaluated using Transwell assays following RASAL2 overexpression or knockdown. Magnification, x200; scale bars=50 µm. Relative cell migration and invasion rates were calculated depending on the number of cells in different fields. (E) BrdU staining was conducted to evaluate the proliferation of RCC cells after RASAL2 overexpression or knockdown. Magnification, x100; scale bars=100 µm ^*P<0.05 vs. Control group. ^#P<0.05 vs. scramble group. OD, optical density; Blank, un-transfected control; Control, control pcDNA.31 plasmid; sh, short hairpin RNA; scramble, scrambled shRNA; RASAL2, ras protein activator like 2; shRASAL2, RASAL2 shRNA.

Figure 5

Effect of manipulating RASAL2 expression on MMP-2, MMP-9 and TIMP-1 expression in RCC cells. Protein expression of MMP-2, MMP-9 and TIMP-1 was assessed by western blotting in ACHN cells following RASAL2 overexpression or knockdown. GAPDH was used as the loading control. ^*P<0.05 vs. Control group. ^#P<0.05 vs. scramble group. Blank, un-transfected control; Control, control pcDNA.31 plasmid; sh, short hairpin RNA; scramble, scrambled shRNA; RASAL2, ras protein activator like 2; shRASAL2, RASAL2 shRNA; MMP, matrix metalloproteinase; TIMP-1, tissue inhibitor of metalloproteinases 1.

Figure 6

Effect of manipulating RASAL2 expression on SOX2 expression, ERK phosphorylation and p38 MAPK phosphorylation in RCC cells. SOX2, ERK, p38 MAPK expression, in addition to ERK and p38 MAPK phosphorylation, were assessed in ACHN cells by western blotting following RASAL2 overexpression or knockdown. GAPDH was used as loading control. ^*P<0.05 vs. Control group. ^#P<0.05 vs. scramble group. Blank, un-transfected control; Control, control pcDNA.31 plasmid; sh, short hairpin RNA; scramble, scrambled shRNA; RASAL2, ras protein activator like 2; shRASAL2, RASAL2 shRNA; p-, phosphorylated.