Down-regulation of estrogen-related receptor alpha (ERRα) inhibits gastric cancer cell migration and invasion in vitro and in vivo

Abstract

Objective: To investigate the correlation between estrogen-related receptor a (ERRα) expression level and gastric cancer (GC).

Methods: We collected GC and adjacent normal tissues from 50 patients. The parameters of the patients were summarized, and correlation with the expression level of ERRα was calculated. Downregulated ERRα using lentivirus was designed and transfected to SGC-7901 and MGC-803 cells. Cell migration, invasion and wound assays were conducted to determine the correlation between ERRα and capacity for cell migration and invasion. The expression level of the genes involved in epithelial-mesenchymal transition, including E-cadherin, γ-catenin, N-cadherin and vimentin, was determined via real-time or quantitative polymerase chain reaction(qPCR) and Western blot analysis.

Results: The expression of ERRα tends to be higher in GC tissues than in adjacent normal tissues. Analyses ofthe expression level of ERRα and patient parameters show that the ERRα level is significantly correlated with TNM staging and patient survival (P<0.05). The downregulation of ERRα can inhibit cell invasion and migration, which was proven by Transwell and cell wound assays. The levels of E-cadherin and γ-catenin increased by conducting qPCR and Western blot analysis. Meanwhile, the levels of N-cadherin and vimentin decreased when ERRα expression was reduced.

Conclusion: ERRα is highly expressed in GC tissues and can promote the migration and invasion of cancer cells. It can be a potential marker for GC diagnosis.

Keywords: epithelial–mesenchymal transition; estrogen-related receptor alpha; gastric cancer.

Figure 1

Immunostaining results of ERRα in GC tissues and adjacent tissues. (A) Immunostaining results of ERRa in GC tissues; (B) Immunostaining results of ERRa in adjacent tissues.

Figure 2

The expression of ERRα in GC tissues and GC cell lines. (A) The expression of ERRa in GC tissues; (B) The expression of ERRa in GC cell lines *P<0.05, **P<0.01.

Figure 3

Kaplan-Meier curves for patients with low ERRα expression versus high ERRα expression. (A) OS curves of patients with low and high expression of ERRα, P=0.022; (B) RFS curves of patients with low and high expression of ERRα, P=0.008.

Figure 4

The cell proliferation and death were measured by CCK-8 (A) and Trypan Blue exclusion assays (B). (A) The cell viability were measured using CCK-8 assay. (B) The death cell population was analyzed using Trypan Blue dye exclusion assay. Data are presented as the mean ± SD. **p < 0.01, *p < 0.05 vs. Vector group.

Figure 5

Low expression of ERRα inhibits the cell migration and invasion. (A) The cell number of migration and invasion in SGC-7901 cell and MGC-803. (B) The migration and invasion ability of SGC-78901 and MGC-803 cells were observed by crystal violet staining **P<0.01.

Figure 6

Cell wound assay indicates the cell migration of the SGC-7901 and MGC-803 cells were inhibited by the low expressed ERRα. (A) Cell wound assay indicates the cell migration of the SGC-7901 was inhibited by the low expressed ERRa; (B) Cell wound assay indicates the cell migration of the MGC-803 was inhibited by the low expressed ERRa.

Figure 7

mRNA level and protein level of the EMT pathway related genes determined by qRT-PCR, western blot and fluorescence staining. (A) mRNA expression level of E-cadherin, γ-catenin, N-cadherin and vimentin; (B) Protein expression level of E-cadherin, γ-catenin, N-cadherin and vimentin. (C) morphology of cells undergoing EMT; (D) Fluorescence staining of E-cadherin, Y-catenin, vimentin, and N-cadherin. *P<0.05.

Figure 8

Knockdown of ERRα inhibits the proliferation of gastric cancer cells in vivo. MGC-803 cells (ERRα and Vector) were subcutaneously inoculated into BALB/c nude mice and tumor growth was monitored. 10 days after tumor inoculation, tumor volume was measured every three days. On the 30th day, mice were sacrificed and tumor xenografts were excised from surrounding tissue and weighted. (A) Representative xenograft tumors for indicated cells were shown. (B) ERRα knockdown significantly reduced xenograft tumor growth in male nude mice by tumor volume examination. (C) ERRα knockdown significantly suppressed xenograft tumor weights. Data represent mean ± S.D, *P < 0.05, **P < 0.05.