Overexpression of Eukaryotic Translation Initiation Factor 5A2 (EIF5A2) Correlates with Cell Aggressiveness and Poor Survival in Gastric Cancer

<div><p>Eukaryotic translation initiation factor 5A2 (EIF5A2) plays an important role in tumor progression and prognosis evaluation. However, little information is available about its potential role in gastric cancer. This study aimed to investigate the function of EIF5A2 in tumor progression and its potential mechanisms. EIF5A2 expression was measured in human gastric cancer cell lines, the immortalized gastric mucosal epithelial cell line (GES-1) and human gastric cancer tissues and knocked down by RNA interference or upregulated by EIF5A2 plasmid transfection. Cell proliferation, migration and invasion were assessed <i>in vitro</i>. The downstream targets of EIF5A2 were examined by western blotting. EIF5A2 and its potential target metastasis-associated protein 1 (MTA1) expression were examined in 160 pairs of human gastric cancer and adjacent non-tumor specimens using immunohistochemistry (IHC) staining, and its correlation with clinicopathological features and survival was investigated. Knockdown of <i>EIF5A2</i> or <i>MTA1</i> caused an apparent suppression of HGC27 cell proliferation, migration and invasion. After knockdown of <i>EIF5A2</i> in HGC27 cells, E-cadherin levels were upregulated and vimentin, cyclin D1, cyclin D3, C-MYC and MTA1 levels were downregulated. Upregulation of EIF5A2 in MKN45 cells resulted in the converse. IHC results showed a positive correlation between EIF5A2 and MTA1 expression in gastric cancers (<i>P</i><0.001). Both EIF5A2 and MTA1 overexpression were correlated with pT stage (<i>P</i>=0.018 and <i>P</i>=0.042), pN stage (<i>P</i>=0.037 and <i>P</i>=0.020) and lymphovascular invasion (<i>P</i>=0.016 and <i>P</i>=0.044). EIF5A2 or MTA1 overexpression was significantly associated with poor overall survival and disease-free survival (All <i>P</i><0.05). Multivariate analyses identified EIF5A2 as an independent predictor for both overall survival (<i>P</i>=0.012) and disease-free survival (<i>P</i>=0.008) in gastric cancer patients. Our findings indicate that EIF5A2 upregulation plays an important oncogenic role in gastric cancer. EIF5A2 may represent a new predictor for poor survival and is a potential therapeutic target for gastric cancer.</p></div

EIF5A2 Expression in human GC tissues and its prognostic significance.

<p>(<b>A</b>) Representative immunohistochemistry images showing EIF5A2 overexpression in moderately-differentiated adenocarcinoma (×200). (<b>B</b>) Overexpression of EIF5A2 in poorly-differentiated gastric adenocarcinoma (×200). (<b>C</b>) Overexpression of EIF5A2 in tumor cells invading vessels (×200). (<b>D</b>) Normal expression of EIF5A2 in non-tumor tissue (×100). (<b>E</b>) The typical staining images showing both EIF5A2 and MTA1 overexpression in the same gastric adenocarcinoma; (<b>G</b>) Overall survival curves for 160 gastric cancer patients receiving gastrectomy, grouped according to EIF5A2 expression (<i>P</i><0.001). (<b>H</b>) Disease-free survival curves for 145 gastric cancer patients receiving curative surgery, grouped according to EIF5A2 expression (<i>P</i> = 0.001).</p

EIF5A2 Expression in GC cells and tissues and validation of the intervention.

<p>(<b>A</b>) <i>EIF5A2</i> mRNA expression was examined by real-time quantitative RT-PCR with <i>GAPDH</i> serving as a loading control. (<b>B</b>) Protein expression in cell lines was confirmed by western blot analysis with GAPDH as a loading control. (<b>C</b>) EIF5A2 protein in human GC tissues was higher than that of the paired distant non-tumor tissues. (D) All three EIF5A2-siRNAs, especially #1, efficiently repressed <i>EIF5A2</i> mRNA expression in HGC27 cells. (E) Western blotting reveals that EIF5A2 was knocked down by the treatment of EIF5A2- siRNAs in HGC27 cells. (<b>F</b>) <i>EIF5A2</i> mRNA was significantly upregulated by transient transfection of EIF5A2 plasmids in MKN45 cells. (<b>G</b>) EIF5A2 protein was significantly upregulated by transient transfection of EIF5A2 plasmids in MKN45 cells. (H and I) T2-siRNA could efficiently suppress MTA1 expression in HGC27 cells.</p

EIF5A2 or MTA1 expression levels influenced the aggressiveness of GC cells in vitro.

<p>(<b>A</b>) Proliferation of EIF5A2-targeted siRNA (T1-siRNA) and non-targeted control (NC) HGC27 cells were measured by CCK-8 assay every 24 h after siRNA transfection. (<b>B</b>) Cell migration and invasion were decreased following transfection of T1-siRNA into HGC27 cells (Student’s <i>t</i>-test, <i>P</i><0.001 and <i>P</i> = 0.001, respectively). Column, mean; bars, ±SD (from triplicates). (<b>C</b>) Proliferation of EIF5A2-plasmid and control plasmid MKN45 cells were measured by CCK-8 assay every 24 h after plasmid transfection. (<b>D</b>) Cell migration and invasion were increased following transient transfection of EIF5A2-plasmid into MKN45 cells (Student’s <i>t</i>-test, <i>P</i> = 2.06×10^-5 and <i>P</i> = 7.65×10^-6, respectively). Column, mean; bars, ±SD (from triplicates). Image insert (B right and D right) shows a representative field of view from each treatment. (<b>E</b>) Proliferation of MTA1-targeted siRNA (T2-siRNA) and non-targeted control (NC2) HGC27 cells were measured by CCK-8 assay every 24 h after siRNA transfection. (<b>F</b>) Cell migration and invasion were decreased following transfection of T2-siRNA into HGC27 cells (Student’s <i>t</i>-test, both <i>P</i> = 0.001). Column, mean; bars, ±SD (from triplicates).</p

Immunoblotting analysis of protein expression after silencing or overexpressing EIF5A2 in GC cells.

<p>(<b>A</b>) After knockdown of EIF5A2 in HGC27 cells, E-cadherin levels were upregulated, while vimentin was downregulated accompanied by cyclin D1, cyclin D3 and C-MYC, and MTA1 downregulation. (<b>B</b>) Ectopic overexpression of EIF5A2 after transient transfection of EIF5A2-plasmid substantially upregulated cyclin D1, cyclin D3, C-MYC and MTA1, accompanied by downregulation of E-cadherin, while vimentin was upregulated.</p

The frequency and percentage for age stratified by gender.

<p>The frequency and percentage for age stratified by gender.</p

Difference Between Measured and Predicted Values Based on the Validation Subset.

*<p>Data are presented as mean of difference (predicted-measured) ±95% CI (confidence interval) of difference, and <i>P</i> value.</p

Predictive Equations Using Regression Analysis for the Developed and other Reference Equations Based on the Training Subset.

<p>Predictive Equations Using Regression Analysis for the Developed and other Reference Equations Based on the Training Subset.</p

Pearson's correlation coefficients between spirometric parameters and height, weight, age in the training subset^*.

*<p><i>P</i><0.001 for all correlation coefficients. FVC, forced vital capacity; FEV1, forced expiratory volume in one second; PEF, peak expiratory flow; MMEF, maximal mid-expiratory flow.</p

Additional file 1: of Medical expenses of urban Chinese patients with stomach cancer during 2002–2011: a hospital-based multicenter retrospective study

Figure S1. Yearly trend of medical expense for stomach cancer diagnosis and treatment per patient, by province groups, 2002-2011 . A to B show the yearly trend of  medical expense per patient  by province (A), by data reliability (B). (PDF 418 kb