The tumor suppressive roles of ARHGAP25 in lung cancer cells

Abstract

Aim: Several Rho GTPase-activating proteins (Rho GAPs) have been proved to serve as tumor suppressors in diverse human cancers. Among them, ARHGAP25 has also been found to be associated with hematopoietic cells and regulate phagocytosis. Little is known about the role of ARHGAP25 in lung cancer cells.

Methods: Quantitative real-time PCR and Western blot were used to measure the expression levels of ARHGAP25. The ability of cell growth and mobility were measured by cell proliferation and Transwell assays. Chromatin immunoprecipitation and luciferase assay were conducted to identify the transcriptional regulation.

Results: Lung cancer tissues had much lower expression level of ARHGAP25 compared to non-cancerous specimens as well as for lung cancer cells. Cell growth and mobility were strongly reduced when ARHGAP25 was overexpressed. Further, significantly negative correlation between ARHGAP25 expression and Wnt signaling pathway was observed. Overexpression of ARHGAP25 reduced the expression of β-catenin and matrix metalloproteinase-7. ARHGAP25 knockdown effect of increased abilities of cell proliferation, migration and invasion could be reversed by adding XAV939 inhibitor. The promoter site of ARHGAP25 could be bound with HOXA4. HOXA4 could regulate the transcriptional activity of ARHGAP25.

Conclusions: This study suggests that ARHGAP25 may inhibit lung cancer cell growth, migration and invasion through Wnt/β-catenin signaling pathway and its transcriptional activity can be regulated by HOXA4.

Keywords: ARHAGP25; HOXA4; Wnt signaling pathway; biological function; lung cancer.

Figure 1

The expression of ARHGAP25 in lung cancer was measured. The difference of ARGFAP25 expression in cancerous and non-cancerous samples was compared in two cohorts (A) n=546; (B) n=60, respectively. (C) Western blot was conducted between paired cancerous and non-cancerous samples (n=6). (D and E) Western blot and qRT-PCR were conducted in four lung cancer cell lines. ***P<0.001.

Figure 2

ARHGAP25 inhibited cell growth. ARHGAP25 expression was evaluated by using RT-PCR (A) and Western blot (B) in NCI-H446 and NCI-H1975 cells in which ARHGAP25 was overexpressed. Wild type group was a control group. Cell growth rate was compared in NCI-H446 and NCI-H1975 cells, respectively (C and D). ***P<0.001.

Figure 3

ARHFAP25 inhibited lung cancer cell, migration and invasion. Transwell invasion assay was performed in NCI-H1299 (A) and NCI-H446 (B) cells in which ARHGAP25 was overexpressed. Wild type group was used as control group. ***P<0.001.

Figure 4

Negative correlation between ARHGAP25 and the Wnt/β-catenin signaling pathway. (A) Gene set enrichment analysis (GSEA) analysis was conducted to display a series of genes negatively associated with Wnt signaling pathway (NES=−2.29, P<0.0001). (B) Western blot was conducted in NCI-H1975 and NCI-H446 cells in which ARHGAP25 was overexpressed. (C and D) qRT-PCR was conducted in NCI-H1975 (C) and NCI-H446 (D) in which ARHGAP25 was overexpressed. Wild type group was used as control group. ***P<0.001.

Figure 5

Role of HOXA4 in the regulation of ARHGAP25 in lung cancer. Western blot (A) and qRT-PCR (B) were conducted in NCI-H1975 and NCI-H446 cells in which HOXA4 was overexpressed. (C) The transcriptional activity of ARHGAP25 promoter was detected by luciferase assay after transfection of HOXA4 siRNA or overexpressed plasmid in NCI-H1299 cells. (D) ARHGAP25 DNA was detected in the chromatin sample immunoprecipitated from NCI-H1299 cells using an antibody against HOXA4. Note: ***P<0.001.

Figure 6

Regulation of HOXA4 on the biological function of ARHGAP25 through the Wnt/β-catenin signaling pathway. Cell proliferation assay (A), Transwell assay (B) and Western blot (C) were conducted in designed groups including vector plus siNC, ARHGAP25 siRNA, siNC plus β-catenin inhibitor XAV939, ARHGAP25 siRNA plus β-catenin inhibitor XAV939, HOXA4 siRNA and HOXA4 siRNA plus ARH OE. ***Indicated significance of siNC (P<0.001); ###indicated significance of siNC plus XAV939 (P<0.001).