Role of the ERas gene in gastric cancer cells

As a novel member of the Ras family, ERas, found in murine embryonic stem (ES) cells in 2003, was considered a pseudogene. To date, there are a few reports on the relationship between ERas and tumors. It was recently suggested that ERas could affect gastric carcinoma (GC) metastasis, but no significant relationship was found with tumor proliferation. Since ERas plays an important role in tumor-like growth of ES cells subcutaneously injected into nude mice, we hypothesized that ERas plays a role in tumor proliferation. In this experiment, we selected 7 GC strains from different sources with different differentiation degrees, we detected the expression of full length ERas transcript, and selected two ERas highly expressing GC strains, MKN-28 and BGC-823. After knocking down the ERas gene by siRNA, we observed that there was a significant decrease in proliferation, metastasis as well as clonality. Therefore, ERas is confirmed to be an important gene in affecting tumor proliferation and metastasis. Furthermore, the significance of the ERas mechanism and signaling pathway is shown.


Introduction
The ERas gene, which can support embryonic stem (ES) cell tumor-like growth (1)(2)(3)(4), was first identified in murine ES cells by Takahashi et al (1). It encodes a protein of 227 amino acids with 43, 46 and 47% identity to conventional Ras oncogenes H-ras, K-ras and N-ras, respectively, defined as a new member of the Ras family. Unlike other Ras family members, the ERas product is a constitutively active protein without any mutation (1), while conventional ras oncogenes acquire activation of carcinogenesis by point mutation of several amino acids, which include Gly12, Ala59 or Glu63 (5,6). The ERas protein contains amino acid residues identical to those present in active mutants of the conventional Ras oncogenes K-ras, N-ras and H-ras (7). The conventional Ras members mainly function through activating either phosphatidylinositol-3-OH kinase (PI3K) or Raf pathway (8,9), while ERas interacts with PI3K (10) but not with Raf (11,12). The human ERas gene was initially erroneously recognized as a processed pseudogene HRasp (Ha-Ras2) (13,14) with deficiency or meaningless mutation, but it was more recently described as a gene potentially encoding a functional human ERas protein (15)(16)(17)(18). Therefore, a few studies on human ERas have previously been reported.
In 2002, Bjorklund et al (19) found that mouse ES cells developed into teratomas when transplanted into nude mice. In 2003, Takahashi et al (1) found that ERas is key in the tumor-like growth properties of ES cells. In 2009, Kaizaki et al (17) discovered ERas was actively expressed in gastric cancer (GC) and was closely related to its oncogenesis. These results indicate that the expression of ERas may be associated with cell proliferation and transformation.
However, in 2010, Kubota et al (20) detected 142 clinical samples of GC, which showed that ERas expression was strongly associated with liver and lymph node metastases. Meanwhile, there was no significant correlation between ERas expression and histological differentiation. Overexpression of ERas in GC cell lines promoted colony formation while it showed no significant effect on cell proliferation. These results indicated that ERas was only associated with the metastasis of GC. However, clonality is an indicator of proliferation capacity. Hence, considering the multiaspect influences of oncogenesis, we decided to knock down the ERas gene in order to study the impactions on proliferation and clonality of GC cells.
In addition, Kameda and Thomson (2) used RT-PCR to analyze expression of the ERas gene in human ES cells in 2005, and they could not detect a full-length ERas coding transcript. Instead, a truncated noncoding transcript was found, which was caused by a premature polyadenylation signal predicted through sequence analysis and confirmed by 3'RACE analysis. Except for the premature PloyA locus, humans and chimpanzees have typical-Alu-s-retrotransposon insertions, which also influence the expression of ERas at this specific locus. Moreover, the lack of ERas expression in human ES cells indicates that the oncogenesis is very different from that of murines (21). These findings indicate that further studies should be performed to ascertain whether or not a fulllength ERas coding transcript is present in human GC cells.
In this study, 8 cell strains from different sources, GC lymph node metastasis, GC liver metastasis, GC ascites and GC tissues, with different differentiation degrees, were chosen to determine whether a full-length ERas mRNA exists and to elucidate the difference of degree of ERas expression among these GC strains by RT-PCR, real-time PCR and western blotting. Furthermore, we confirmed the effect of ERas knockdown on cell proliferation, metastasis and clonality in ERas highly expressed GC strains. Amplification of the ERas full-length transcripts and sequencing analysis. Total cellular RNA was extracted from each cell line with TRIzol ® (Invitrogen Life Technologies, Carlsbad, CA, USA), and cDNA was synthesized with Reverse Transcription kit (Promega Corp., Madison, WI, USA), and both were performed according to the manufacturer's protocol. The cDNA was synthesized by PCR with PrimeSTAR HS DNA polymerase (Takara Bio, Shiga, Japan). The primers used for the full-length ERas coding sequence were: forward, 5'-atggagctgccaacaaagcctggca-3' and reverse, 5'-ttcaggccacagagcagccacagt-3', which gave an amplified fragment of 702 bp. The reaction conditions were: 94˚C for 15 sec, 62˚C for 45 sec and 72˚C for 30 sec, repeated for 35 cycles. The products were separated by 1.2% agarose gel electrophoresis, and the 702 bp bands were recycled using aqua-Spin gel extraction mini kit (Watson Biotechnologies, Inc., Shanghai, China). The identification of PCR products was confirmed by sequencing analysis (Songon Biotech Co., Shanghai, China).
Western blot analysis. Cells were lysed in a lysis buffer containing 2% sodium dodecyl sulfate (SDS) and 0.125 M Tris-HCl (pH 6.8) on ice for 30 min, followed by high-speed centrifugation, and the supernatant protein was finally collected. SDS-PAGE was performed using 10% polyacrylamide gels. PAGE separated proteins were electrophoretically transferred onto nitrocellulose membranes. The membrane filters were blocked with 5% powdered milk in TBST (0.1% Tween-20) for 2 h and then incubated in rabbit ERas antibody (Abgent, Suzhou, China) diluted 1:100 in TBST at 4˚C overnight, and finally incubated with HRP anti-rabbit secondary antibody (Kangchen Biotech, Shanghai, China) diluted 1:2,000 for 1 h at room temperature. Antigens on the membrane were detected with enhanced chemiluminescense detection reagents (Roche, Basel, Switzerland).
CCK-8 assay. siRNA-transfected MKN-28 and BGC-823 cells were seeded into 96-well plates at a density of 3x10 3 cells/well and maintained in culture medium for 5 days. Each well set five duplicates. We measured cell growth using cholecystokinin (CCK) assay by Cell Counting Kit (Dojindo, Tokyo, Japan) according to the manufacturer's instructions.
Cell migration assay. For wound-healing experiments, MKN-28 and BGC-823 cells transfected with siRNA were cultured to 80% confluence after being seeded into 6-well plates, then scraped using a p10 tip (time 0), and suspended cells were washed with PBS three times. Cells were incubated for another 4 days and images were captured by microscope (Zeiss, Oberkochen, German) at the same time every day. Migration distance was measured from images (5 fields) at each indicated time point.
Transwell assay of MKN-28 and BGC-823 cells was assessed using 6.5 mm diameter inserts (Corning Costar Corp., Corning, NY, USA). A total of 3x10 4 cells were suspended in 100 µl serum-free RPMI-1640 medium and loaded into upper wells; lower chambers were filled with 600 µl of complete medium (RPMI-1640 supplemented with 10% FBS). Migration chambers were incubated in a humidified 5% CO 2 incubator at 37˚C for 24 and 48 h. Cells were then fixed with 600 µl of paraformaldehyde for 20 min. The inner surfaces of the upper chambers were wiped using cotton swabs to remove nonmigrated cells in the migration assay. The chambers were then washed with PBS and stained with 500 µl crystal violet for 20 min at room temperature. Stained cells were counted using the ImageJ software, and 5 random fields were counted (Zeiss).
Colony formation assay. A total of 500 siRNA-transfected MKN-28 and BGC-823 cells were seeded in 6-well plates and incubated for 14 days respectively, with the medium replaced every 4 days. On the 15th day, the cells were stained with crystal violet for 20 min and washed with tap water for 10 min. For each dish, colonies in five random fields were counted using the ImageJ software.
Statistical analysis. Each measurement was performed in triplicate. Original real-time PCR data, CCK-8 data, migration/invasion data and colony formation data were recorded as continuous variables and analyzed using Student's t-test or linear polynomial ANOVA with LSD post hoc examination. All statistical analyses were performed using SPSS 16.0 software. P-values <0.05 were considered to indicate statistically significant differences.

Results
ERas expressed in GC cells. The full-length ERas mRNA transcript was detected in all seven GC cell lines and GES-1 gastric mucosa cell line by RT-PCR. The forward and reverse primers were located in open reading frame (ORF) 1-25 and 680-702 bp separately, which give rise to an amplified fragment of 702 bp (Fig. 1A). To assess whether ERas expression was different with mutant H-ras, K-ras and N-ras, mutation analysis was performed by sequencing analysis, revealing no mutation of ERas in all the gastric cell lines that we selected (data not shown).

ERas increases GC cell proliferation.
To examine the role of ERas in cell proliferation, we measured BGC-823, MKN-28 cell growth by CCK-8 assay after transfecting with ERas siRNA30 and siRNA32.
We observed a significant decrease in cell proliferation when the expression of ERas was knocked down in MKN-28 and BGC-823 cells (Fig. 2).
On the third day, the OD value of MKN-28 cells decreased from 1.60±0.05 to 1.28±0.10 when transfected with siRNA30  ERas promotes GC cell migration. We also confirmed the effect of ERas on migration in MKN-28 and BGC-823 cells by Transwell and wound-healing assay.
As shown in Fig. 3A and B, knockdown of ERas by siRNA significantly impaired the ability of MKN-28 and BGC-823 cells to migrate through the membranes. Twenty-four hours later, the number of migratory cells decreased from 163.5±9.19 Then, we further analyzed migration ability by using wound-healing for 4 days. As shown in Fig. 4, the speed of wound repair was markedly slower when ERas was silenced by siRNA in MKN-28 and BGC-823 cells at 24, 48, 72 and 96 h. The statistical significance was most notable at 48 h, when the wound repair percentage decreased from 61.35±3.54% to 25±6.25% (P<0.001) and 28.89±3.85% (P<0.001) after ERas was knocked down by siRNA30 and siRNA32 respectively in MKN-28 cells, while the percentage decreased from 58.13±3.61% to 19.61±3.78% (P<0.001) and 18±3.85% (P<0.001) respectively in BGC-823 cells.

Discussion
The ERas gene is strongly expressed in BGC-823 and MKN-28 cell strains among the eight gastric cell lines (Fig. 1B-D). To examine the effect of ERas on GC cell proliferation, these two highly expressing endogenous ERas cell strains were investigated after treatment with ERas siRNA by CCK-8 assay for 5 days. Knockdown of ERas inhibited the proliferation ability markedly compared to control in BGC-823 and MKN-28 cells on the third and fourth day (Fig. 2). Kubota et al (20) concluded that ERas could not promote the proliferation in GCIY cells transfected with an ERas-overexpressing vector by MTS assays for 6 days, whereas it could enhance colony formation as the cell clonality experiment results showed. They came to the same result that there was no relationship between proliferation and the ERas gene in GCIY and NUGC-4 cells with ERas knocked down by stealth siRNA using MTS assays for 2 days. However, whether or not knockdown of ERas inhibits cell colony formation was not further established. GCIY cell strain was used in both ERas overexpression and knockdown experiment. However, the expression level of ERas in this strain was relatively low, indicating it was not suitable for the knockdown experiment. On the other hand, the observation period in their study was relatively short. These reasons may lead to the disparity. Meanwhile, the results of Transwell test, scratch test and colony formation test (22,23) proved that ERas is able to increase GC cell migration and colony formation which were also two aspects that could reflect cell proliferation. Hence, there is sufficient evidence to prove that ERas enhances GC cell proliferation.
Furthermore, ERas was expressed most highly in poorly differentiated BGC-823 cells and well differentiated MKN-28 cells, less highly in poorly differentiated SNU-16 cells, moderately differentiated SGC-7901 cells and poorly differentiated MKN-45 cells, almost silently in poorly differentiated AGC cells, immortal GES-1 cells and poorly differentiated NCL-N87 cells (Fig. 1A-C). From these date, the conclusion that expression of ERas is not related to histological differentiation is the same as that of Kubota et al. However, from analyzing the following seven cell strains, SGC-7901 from GC lymph metastasis, NCL-N87 from GC liver metastasis, SNU-16 from GC ascites, GES-1 from fetal gastric mucosa and others from GC tissues, the connection between ERas and metastasis of gastric lymph and liver is not so strong, which is different from the result of Kubota et al. Therefore, to fully understand the connection between ERas and gastric lymph metastasis and liver metastasis, further research is required.
The full-length transcript of ERas (2,20) in those 7 GC cell strains were examined (Fig. 1A), suggesting that activated ERas is present universally in GC cells. Since a full-length transcript cannot be found in human ES cells, there should be some common activation factors which inhibit the prematuration of early polyadenylation signal and the insertion of Alu-S transposons to activate ERas expression.
To date, there are few studies on the relationship between ERas and cancer, and the function of ERas has yet to be determined. This study used CCK-8, Transwell, scratch test and clonality test to successfully prove that ERas has the ability to enhance GC cell proliferation, metastasis and clonality. Compared to Kubota et al (20), this study proved that the Figure 5. The effect of ERas on colony formation ability. After 24 h of siRNA30, siRNA32, negative control transfection, 5x10 2 cells were transferred into 6-well plates and incubated for 14 days. The cells were stained with crystal violet, photographed by a microscope (x50 magnification; Zeiss) and an ordinary camera. Bar charts show the number of colonies; significances were analyzed by using the Student's t-test ( ** P<0.01 vs. control).
activation of the ERas gene in GC cells is common and the function of ERas in the processes of GC cell development and metastasis is important; there must be a significance of clarifying the correlative key signal pathways and the cytokines to activate the pathways.