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

Expression levels of ERRα in GC

The expression of ERRα was determined via tissue array. We collected 50 cases of GC and adjacent tissues for detection. Immunohistochemical results showed that the expression of ERRα in GC tissues was higher than that in adjacent tissues (Figure 1A, 1B). The mRNA expression level of ERRα was 0.97±0.27 in GC tissues and 0.74±0.25 in adjacent tissues (Figure 2A). Analysis showed that the expression level of ERRα in GC tissues was significantly higher than that in adjacent tissues (P<0.01). A high expression of ERRα was also identified in the five GC cell lines (AGS, SGC-7901, MGC-803, BGC-823 andHGC-27) compared with that in GES1 (P<0.05, Figure 2B). We categorised tissues as ERRα high or low expression the basis of the median. Amongst the GC tissues, 26 had high ERRα expression and 24 had low expression.

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.

Clinical value of ERRα expression for patients with GC

To study the clinical significance of ERRα in GC, we analysed the clinical relationship between the expression of ERRα in patients with GC and clinicopathological characteristics. The results showed that a high expression of ERRα was associated with advanced TNM stage (Table 1, P=0.002). Furthermore, the Kaplan–Meier analysis indicated that patients with high ERRα expression exhibited a decrease in OS (Figure 3A, P=0.022) and relapse-free survival (Figure 3B, P=0.022). The multivariate analysis results showed that a high expression of ERRα was a significant independent prognostic factor for OS in patients with GC (Table 2, P=0.024).

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.

Downregulation of ERRα inhibited proliferation, invasion and migration of GC Cells

SGC-7901 and MGC-803 cell lines were used to perform cell proliferation, migration and invasion assays. The ERRα vector was down regulated for transfection to downregulate the ERRα level in cells. An empty vector was used as control. Western blot analysis was conducted to determine the ERRα level in cells. The results showed that the low expression of the ERRα vector evidently decreased the expression of ERRα in SGC-7901 and MGC-803 cells compared with the empty vector (data not show). Therefore, the vector was successfully transfected into the cells. The trypan blue rejection method and CCK-8 assay were used to measure the cell proliferation and viability. The results showed that the knocking down ERRα can significantly inhibit proliferation capabilities without affecting the cell death (Figure 4). The cells transfected with the vectors were then used for cell Transwell migration and invasion assays. Cells that migrated to or invaded the bottom chamber were stained and counted. The results indicated that the downregulation of ERRαsignificantly attenuated the capacity for cell migration and invasion in SGC-7901 and MGC-803 cells (Figure 5A, 5B).

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.

Wound scratch assay

We also performed cell wound scratch assay. The wound was recorded at 0, 24 and 48h after it was generated. The downregulation of ERRα can weaken cell migration significantly in SGC-7901 and MGC-803 cells, particularly MGC-803 cells (Figure 6A, 6B).

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.

Target gene of ERRα

To verify the mechanism of ERRα in regulating cell migration and invasion, qPCR and Western blot analysis were conducted to determine the mRNA and protein levels of E-cadherin, γ-catenin, N-cadherin and vimentin, which are involved in the epithelial–mesenchymal transition (EMT) signaling pathway. The results showed that the mRNA and protein levels of E-cadherin and γ-catenin were increased, while the levels of vimentin and N-cadherin decreased in both cell lines (Figure 7A, 7B), indicating that the EMT signaling pathway was inhibited and potentially improved cell migration and invasion when ERRα had a low expression. To further illustrate the EMT stage cells, we performed immunofluorescence staining to more closely observe the cell morphology and differentiation occurred during EMT. As shown in Figure 7C, the cell density of cells with ERRα downregulated is lower than control. The immunofluorescence staining of cells presents consistent result with western blot, and the cells in control group looks less edged.

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.

Downregulation of ERRα inhibited the growth of GC cells in vivo

To assess the antitumor activity of ERRα in vivo, MGC-803 cells (shERRα and vector) were subcutaneously inoculated into BALB/c nude mice and tumor growth was monitored. As shown in Figure 8A, 8B, the knockdown of ERRα can significantly inhibit tumor growth compared with the vector. The knockdown of ERRα significantly decreased tumor volume and weight (Figure 8B, 8C, P<0.05; Figure 5B, P<0.05). Overall, these findings indicated that knocking down ERRα can suppress GC cell growth in vivo.

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.

Collection of tissue specimens

GC and adjacent tissues were collected from 50 patients diagnosed in Jiangsu Cancer Hospital from 2017 to 2019. The samples were immediately fixed in 10% formalin solution for 14 h. Then, paraffin embedment was performed for further immunohistochemical study. Pathological stage was determined on the basis of Fuhrman nuclear grade. Tumor stage was determined in accordance with the TNM classification of the American Joint Committee on Cancer. Prognosis assay was performed to follow-up with survivors and estimate the overall survival (OS) rate. OS was calculated from the date of surgery to the date of death or the latest follow-up. The present study was approved by the Ethics Committee of Jiangsu Cancer Hospital. All the participants provided a signed informed written consent in advance.

Tissue array and immunohistochemistry staining

Human GC tissue microarray was constructed using formalin-fixed and paraffin-embedded kidney tissues. Immunohistochemical studies were performed using a Leica autostainer XL ST5010 (Leica Biosystems, Wetzlar, Germany). The sections were dehydrated with ethanol and then subjected to antigen retrieval in citrate buffer (10 mM, pH 6.0) for 30 min. The sections were blocked in peroxidase-blocking reagent (Dako Cytomation, Glostrup, Denmark) for 15 min at room temperature, followed by incubation with rabbit anti-human polyclonal primary antibody (1:1000 dilution; Abcam, Cambridge, UK) overnight at 4° C in a humidified chamber. Sections were then incubated with mouse anti-rabbit secondary antibody (1:1500 dilution; Abcam, Cambridge, UK) for 30 min at RT. After washing completely with phosphate-buffered saline (PBS), the sections were developed in freshly prepared diaminobenzidine solution, counterstained with haematoxylin, dehydrated through graded ethanol, cleared with xylene and cover-slipped. The expression of ERRa was estimated in accordance with the percentage of stained cells. Each sample was classified either low-expression (≤30%) or high-expression (>30%).

Cell culture

GC cells (AGS, SGC-7901, MGC-803, BGC-823and HGC-27) and human normal gastric epithelial cell (GES1) were obtained from the Shanghai Cell Bank (Shanghai, China) and cultured at 33° C with 10% CO₂. Dulbecco’s modified Eagle’s medium (DMEM) in high glucose with 10% fetal bovine serum (FBS; Gibco, USA) was supplemented. Cells were cultured, amplified and passaged. After 3 days, the cells were digested and pelleted via centrifugation. Cell morphology was observed using a light microscope, and the cells were suspended to a concentration of 1×10⁶/mL.

Cell transfection

The lentiviral shRNA expression vector targeting shERRα and scrambled control were obtained from Obio Technology (Shanghai, China). Cells were seeded into six-well plates at a density of 2×10⁵ cells/well. After 80% confluence was reached, shERRα (ERRα)and scrambled control siRNA (vector) were transfected into cells. After 48 h, real-time or quantitative polymerase chain reaction(qPCR) and Western blot analysis were performed to determine transfection efficiency.

qPCR

The mRNA level of ERRα was verified via qPCR. M-MLV reverse transcriptase (Promega, USA) was used to synthesise cDNA. PCR was prepared with GoTaq qPCR Master Mix (Promega, USA) and performed on an ABI 7500 system (Applied Biosystem, USA). GAPDH was used as housekeeping gene to perform comparative quantification.

Western blot analysis

Cells were lysed in 1% sodium dodecyl sulphate (SDS) lysis buffer. Bicinchoninic acid (BCA) assay was performed to determine protein concentration. SDS-polyacrylamide gel electrophoresis (10%) was used to separate the protein. The protein was then transferred to a nitrocellulose membrane. Nonfat milk in PBS was used to block the membrane at room temperature for 1h. The membrane was incubated overnight at 4° C with primary antibody (Abcam, UK). After washing several times with PBS, the membrane was incubated in blocking buffer with a secondary antibody coupled to horseradish peroxidase for 2h at room temperature. Complexes were formed on the membrane, and the membrane was detected using ECL Plus (Amersham Biosciences/GE Healthcare, Velizy, France).

Migration and invasion assays

A 24-well plate containing 8mmpore size chamber inserts (Corning, USA) was used to evaluate the migration and invasion of tumor cells. For the migration assay, 1×10⁵ cells were seeded into the upper chamber. For the invasion assay, the membrane was coated with Matrigel (BD Biosciences, USA) to form a matrix barrier, and then 2×10⁵ cells were placed in the upper chamber. In each lower chamber, 600mLof DMEM with 10% FBS was added. Cells were incubated at 37° C and allowed to migrate for 36h or invade for 48h. After incubation, the cells that migrated through the pores were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Then, the cells were counted and photographed under an IX71 inverted microscope (Olympus, Tokyo, Japan).

Wound scratch assay

The cells were seeded into 12-well plates, cultured to confluence and wounded by scraping with a 0.1–10 μL pipette tip. The cells were washed with PBS. The cell culture was then re-fed with the medium for 24h or 48h. The cells were then fixed with formaldehyde and stained with 0.1% toluidine blue at room temperature for 30min. Images of the cells were captured using a camera-equipped microscope (Carl Zeiss, USA).

In vivo tumorigenesis assay

Male BALB/c nu/nu mice (4–5 weeks old) purchased from the Laboratory Animal Centre of Shanghai, Chinese Academy of Sciences (Shanghai, China), were housed under specific pathogen-free conditions. The mice were randomly divided into two groups (5 mice/ group). MGC-803 cells (shERRα and vector) were subcutaneously inoculated into the BALB/c nude mice, and tumor growth was monitored. Viable cells (3×10⁶ cells/group) were injected subcutaneously into the flanks of the mice. Then, 10 days after cell injection, the length (L) and width (W) of tumor xenografts were measured with a Vernier calliper at four- or three-day intervals. Tumor volumes were calculated (V = W² × L/2). The animals were sacrificed under general anaesthesia with chloral hydrate (5%, 100 μL/10 g).

Statistical analysis

The association between ERRα expression and pathological parameters was calculated via the Chi-squared test. ERRα expression between normal and GC samples was compared using Student’s two-tailed t-test. The correlation between the two groups of samples was evaluated using the Pearson correlation coefficient. The correlation between the ERRα expression and pathological indexes of the patients was calculated using Spearman’s correlation analysis. The Kaplan–Meier method was applied to analyse patient survival, and the log-rank test was adopted to estimate differences between groups. Overall, survival was defined as the interval from the date of initial surgery to the date of death or last contact. Patients who lost contact were treated as survivors. The Cox proportional hazards regression model was used for multivariate analysis to study the contribution of various potential prognostic parameters to OS.

All the data were presented as mean ± standard error. Significance was established as P<0.05. All the analyses were performed using SPSS 22.0 software.

Ethics approval and consent to participate

The study was approved by the Ethics Committee of the Department of thoracic surgery of Jiangsu Cancer Hospital and written informed consent was obtained from each participant.

Availability of data and materials

All of the data supporting our findings can be found in the main paper. All data of the data generated in this study are available upon request by writing to the corresponding author.