Astrocyte Elevated Gene 1 (AEG-1) Acts as a Promoter Gene in Clear Cell Renal Cell Carcinoma Cell Growth and Metastasis

Abstract

BACKGROUND Clear cell renal cell carcinoma (ccRCC) is usually incurable once it progresses to metastatic stage. Hence, in-depth investigations to reveal the precise molecular mechanisms behind the metastasis of ccRCC are required to improve the therapeutic outcome of ccRCC. MATERIAL AND METHODS The level of astrocyte elevated gene 1 (AEG-1) in ccRCC tissues and cell lines was determined by quantitative real-time PCR (qRT-PCR) assay. The MTS, colony formation, wound-healing, and Transwell invasion assays were used to assess the role of AEG-1 in ccRCC cells growth, migration, and invasion in vitro, respectively. Xenograft model and lung metastasis models were constructed to analyze the functions of AEG-1 in ccRCC cells growth and metastasis in vivo. RESULTS We found that AEG-1 was overexpressed in ccRCC and was associated with the progression of ccRCC. Knocked-down AEG-1 impaired the migration and invasion of ccRCC cells in vitro. Furthermore, under-expression of AEG-1 caused complete inhibition of ccRCC cells growth and metastasis in vivo. In contrast, overexpression of AEG-1 significantly increased the migration and invasion ability of ccRCC cells in vitro. Finally, we revealed that AEG-1 boosted the metastatic ability of ccRCC cells via regulating Notch homolog 1 (Notch1). CONCLUSIONS The AEG-1/Notch1 signaling axis plays a vital role in ccRCC cell growth and metastasis.

Figure 1

Aberrant overexpression of AEG-1 in ccRCC. (A) Representative image of H&E staining of ccRCC tissues. Representative images of IHC staining with AEG-1 in ccRCC tissues. Scale bars: 100 μm. (B) The average of mean optical density (MOD) of AEG-1 staining in the carcinoma tissues (n=50) and adjacent tissues (n=50). ** P<0.01 compared with normal tissues. (C) AEG-1 IHC staining scores in the cancer tissues (n=50) in different AJCC stages. AJCC stages: I–III. ** P<0.01 compared with stage I. (D) The mRNA level of AEG-1 in ccRCC cell lines and an immortalized proximal tubule epithelial cell line, HK-2 was determined by qRT-PCR. The relative AEG-1 mRNA expression level was normalized to U6. ** P<0.01 compared with HK-2 cells. (E, F) Box plots show increased levels of AEG-1 in ccRCC (right) compared with normal kidney tissues (left).

Figure 2

The effect of shAEG-1 on ccRCC cells metastasis. (A) The level of AEG-1 in Caki-2 or 786-O cells and cells transfected with shAEG-1 was detected by qPCR and immunoblotting. (B) Wound-healing assay was used to determine the migration of cells. Scale bar represents 200 μm. (C) Caki-2 or 786-O cells were transfected with shAEG-1 and were subjected to Transwell invasion assay. Scale bar represents 100 μm. (D) MTS assay was applied to analyze cell proliferation. (E) Mean number of tumor colonies formed by AEG-1-shRNA-transfected cells and control ccRCC cells. ** P<0.01 compared with control cells.

Figure 3

Confirmation the role of AEG-1 in ccRCC metastasis. (A) The levels of AEG-1 in vector-control transfected cells or AEG-1-overexpressing ccRCC cells were examined by qRT-PCR and Western blotting. (B) The cells migration was determined using wound-healing assay. Scale bar represents 200 μm. (C) Cell invasion was analyzed using Transwell invasion assay. Scale bar represents 100 μm. (D) Representative images of lung from nude mice after 4 weeks of injection with AEG-1-overexpressing ccRCC cells or shAEG-1 ccRCC cells. Numbers of lung metastasis lesions were quantified. ** P<0.01, compared to control.

Figure 4

AEG-1 increases the metastasis of ccRCC cells via regulating Ntoch1. (A) AEG-1 knock-down altered the expression of pro-metastasis factors in shAEG-1-transfected Caki-2 cells compared to control cells. Gene expression analysis was performed using qRT-PCR. (B) The levels of Notch1 in Caki-2 cells were detected by immunofluorescence. (C) The level of Notch1 was assessed by qRT-PCR. (D) Wound-healing assay was used to analyze the migration of cells that transfected shAEG-1 alone or shAEG-1 combination with pCLEN-Notch1 plasmid. Scale bar represents 200 μm. (E) Transwell invasion was used to identify the invasion of cells. ** P<0.01 compared to control cells, ^## P<0.01 compared to cells transfected with shAEG-1 alone. Scale bar represents 100 μm. (F) After being treated with FLI-06, the level of Notch1 was analyzed by qRT-PCR assay. (G) Wound-healing assay was used to evaluate the migration of AEG-1 overexpressing cells that were treated with FLI-06. Scale bar represents 200 μm. (H) Transwell invasion assay was used to evaluate the invasion ability of AEG-1-overexpressing cells treated with FLI-06. Scale bar represents 100 μm. ** P<0.01 compared to cells transfected with vector, ^## P<0.01 compared to cells transfected with AEG-1.

Figure 5

Tumorigenicity of AEG-1 in vivo. (A) Following subcutaneous injections of Caki-2 cells in athymic nude mice and tumor growth for 35 days, photographs of the tumors were obtained at necropsy. The volumes of the generated tumors were measured once a week. (B) Mice were killed 35 days after the subcutaneous injection. Scatter plot analysis of the mouse tumor weights. (C, D) IHC staining of AEG-1 and Notch1 in the tumor mass from each group. ** P<0.01 compared to scramble.