| Objective: Lung cancer remains the leading cause of cancer-related death worldwide, mainly because it is often diagnosed at advanced stage accompanied by extensive invasion and metastasis. Therefore, investigations of the molecular mechanisms underlying progression and metastasis of non-small-cell lung cancer(NSCLC) may help develop novel therapeutic targets for the malignancy. Our previous data showed that Pim-1 kinase was a proto-oncogene and its dysregulation contributed to tumorigenesis and progression of NSCLC. However, the reason that Pim-1 was upregulated in NSCLC remained to be determined. Eukaryotic initiation factor 4E(e IF4E) was considered as the rate-limiting component for initiation of cap-dependent m RNA translation. EIF4 E overexpression could cause preferential translation of m RNAs, like growth promoting protein and oncogenic proteins. Whether e IF4 E is involved in the regulation of Pim-1 expression and tumorigenesis of NSCLC remained uncertain. Therefore, in the present study, we aimed to evaluate the roles of e IF4 E in tumorigenesis and drug resistance of NSCLC, and to explore the regulatory mechanism of e IF4 E on Pim-1 expression in lung tumorigenesis.Methods:1 Immunohistochemical study(IHC)Expressions of e IF4 E and Pim-1 protein were detected in formalin fixed and paraffin-embedded(FFPE) sections of NSCLCs by immunohistochemical studies(ElivisionTMplus three-step method). The clinicopathologic significance of e IF4 E and Pim-1, and the relationship between the two proteins were evaluated further.2 Cell cultureA549, H1299, H358, H157 and H460 human lung cancer cell lines were maintained in RPMI-1640 medium containing 10% fetal bovine serum and penicillin/streptomycin. Cultures were maintained in a 5% CO2 humidified atmosphere at 37°C. Cells in logarithmic growth phase are digested with 0.25% trypsin and 0.02% EDTA, and used for our study.3 si RNA transfectionTransfections were performed using Lipofectamine 2000 reagent(Invitrogen) following the manufacturer’s protocol. Cells were transfected with Pim-1 or e IF4 E si RNA(100 pmol) or their relative control sequences. Cells were harvested 48 hours after transfection and used for the in vitro test.4 MTT assayCell viability was assessed at 0, 24, 48, 72 and 96 h after e IF4 E si RNA transfection in H1299 and A549 cells using MTT assasy. For drug treatment, cell growth was assessed at 48 h after different concentration of gefitinib treatment, and the IC50 value of gefitinib was calculated in H1299 and A549 cells, respectively. Furthermore, cell viability was evaluated after si-Pim-1 or si-e IF4 E transfection combined with gefitinib by MTT assay.5 Western BlottingTotal proteins(50-100μg) extracted from H1299 and A549 cells 48 h after transfection were analyzed by SDS-polyacrylamid gel electrophoresis and were transferred electrophoretically to nitrocellulose membrane. To evaluate expression of Pim-1 and e IF4 E, blots were blocked with 5% nonfat milk in Tris-Buffered Saline and Tween 20, and incubated with a primary rabbit monoclonal antibody Pim-1 or e IF4 E. Antibody for β-actin was used as a control. The blots were visualized by the chemiluminescence and scanned using Image Quant LAS 4010 Imaging System.6 Real-time PCRExpressions of Pim-1 and eif4 E at m RNA level were determined in H1299 and A549 cells 48 h post transfection by using Real-time PCR. β-actin was used as an internal control. The ratio of target gene is calculated by the method of Comparative Ct(ΔΔCt).7 Statistical analysisSPSS17.0 software was used in our study. Data are presented as mean ± SD. Statistical comparisons between experimental groups were analyzed by t test and χ2 test. Spearman’s correlation analysis was used to determine correlation between Pim-1, e IF4 E expression level, and clinical characteristics of the NSCLC patients. P<0.05 was taken to indicate statistical significance.Results: 1 Expression of e IF4 E and its association with the clinicopathological parameters of NSCLC patientsImmunohistochemistry results showed that the positive staining for e IF4 E was found in the cytoplasm of the tumor cells. EIF4 E staining was found in 56 of 69 NSCLCs, the positive expression rate was 81.1%(56/69), occurred 37 of 41 adenocarcinoma(90.2%), 16 of 23 squamous cell carcinoma(69.6%) and 3 of 5 other types(60.0%). EIF4 E expression was significantly associated with tumor diameter(P=0.036). However, there were no significant associations between e IF4 E expression and other important clinical pathological features(P>0.05). 2 Effects of e IF4 E on cell growth and migration in NSCLC cells in vitro 2.1 Knockdown efficiency of e IF4 E si RNA transfectionA549 and H1299 cells which had higher e IF4 E expression were transfected with si RNA specific for e IF4 E. 48 h after transfection, e IF4 E expression at m RNA and protein level was detected in A549 and H1299 cells by Real-time PCR and Western Blot method, respectively. In comparison with control group, e IF4 E m RNA expression was decreased by 83% and 80% in A549 cells and H1299 cells, respectively. Consistant with Real-time PCR results, e IF4 E protein was inhibited obviously by e IF4 E si RNA compared to the control group in the two cell lines, which suggested that the e IF4 E si RNA sequences could inhibit e IF4 E expression effectively. 2.2 Effects of e IF4 E si RNA transfection on cell growthMTT result showed that cell growth was significantly suppressed in cells transfected with si-e IF4 E compared with the control cells at 48 h, 72 h and 96 h after transfection both in A549 and H1299 cells(P<0.05). Furthermore, cell survival rate was significantly decreased with time-increasing. The results indicated that knockdown e IF4 E expression could inhibit A549 and H1299 cell growth. 2.3 Effects of e IF4 E si RNA transfection on cell migrationMonolayer wound healing assay was performed to observe the role of e IF4 E si RNA transfection in A549 and H1299 cells. The results showed that the speed which cells migrated towards the scratch was lower in e IF4 E si RNA transfected cells when compared with the control ones 48 h after the scratch, indicated that loss of e IF4 E considerably suppressed the migratory ability of the NSCLC cells. 3 Effects of e IF4 E on Pim-1 expression in A549 and H1299 cellsTo explore the effects of e IF4 E on the regulation of Pim-1 expression and tumorigenesis of NSCLC, we knocked down e IF4 E expression by si RNA and determined its impact on Pim-1 expression. In comparison with control cells, reduced levels of Pim-1 protein were detected in si-e IF4 E transfected A549 and H1299 cells. However, there was no change on Pim-1 m RNA expression after si-e IF4 E transfection in both of the cells. The results suggest that e IF4 E expression controls the synthesis of Pim-1 protein in NSCLC cells. 4 Relativity of protein expressions of e IF4 E and Pim-1 in NSCLC 4.1 Expression of Pim-1 and its association with the clinicopathological parameters of NSCLC patientsImmunohistochemistry results showed that the positive Pim-1 staining was detected in the nucleus and cytoplasm of tumor cells. Positive staining of Pim-1was found in 66.2%(51/77) of NSCLC tissues, but only 14.3%(3/21) in normal lung tissues. Furthermore, Pim-1 expression was significantly higher in NSCLC tissues than in adjacent normal tissues(P<0.001). In addition, Pim-1 expression in NSCLC tissues was significantly associated with tumor size, lymph node metastasis, histological type and clinical staging(P<0.05) in NSCLC patients. 4.2 The correlation analysis between Pim-1 and eif4 E protein expression in NSCLC patientsAmong 69 NSCLC samples, 62.3%(43/69) of tumors positive on e IF4 E were also Pim-1 positive, and 15.9%(11/69) e IF4 E negative tumors were Pim-1 negative. There was a positive correlation between e IF4 E and Pim-1 expressions in the specimens(r = 0.504, P<0.001). 5 Effects of Pim-1or eif4 E si RNA transfection combined with gefitinib on cell growth in NSCLC cells 5.1 The growth curve of A549 and H1299 cells after gefitinib exposureMTT assay was used to define the IC50 value of gefitinib exposure in A549 and H1299 cells. The results showed that the IC50 in A549 and H1299 was 10.56μM and 20.17μM, respectively. Based on these data, A549 and H1299 cells were exposed to 10μM and 20μM gefitinib in our study. 5.2 The effects of e IF4 E si RNA transfection combined with gefitinib on cell survivalCell survival rates were detected in A549 and H1299 NSCLC cells transfected with control si RNA or si-e IF4 E combined with drug treatment using MTT assays. Inhibition of e IF4 E expression by e IF4 E si RNA was able to suppress survival of A549 cells at 72 h after gefitinib exposure(P<0.05). Furthermore, the viability of H1299 cells was decreased significantly upon si-e IF4 E in combination with gefitinib treatment compared with the control cells at 48 and 72 h. The above results suggested that knockdown e IF4 E expression could increase the sensitivity of NSCLC cells to gefitinib. 5.3 The effects of Pim-1 si RNA combined with gefitinib treatment on cell survivalMTT assay results showed that knockdown Pim-1 expression by Pim-1 si RNA was able to suppress survival of A549 cells at 48 h and 72 h after gefitinib exposure(P<0.05). In addition, the viability of H1299 cells was decreased significantly upon si-Pim-1 in combination with gefitinib treatment compared with the control cells at 72h(P<0.05). The above results suggested that knockdown Pim-1 expression could increase the sensitivity of NSCLC cells to gefitinib.Conclusion:1 EIF4 E was frequently overexpressed in human NSCLCs, and its expression was associated with tumor size significantly. Knockdown e IF4 E expression could inhibit the ability of cell growth and migration in NSCLC cells in vitro.2 Pim-1 was frequently overexpressed in NSCLCs, and its expression was associated with tumor size, lymph node metastasis, histological type and clinical staging.3 There was a positive correlation between e IF4 E and Pim-1 protein expression in human NSCLCs, and e IF4 E could regulate the synthesis of Pim-1 protein in NSCLC cells.4 Pim-1 si RNA or e IF4 E si RNA transfection could increase the sensitivity of EGFR-TKI gefitinib. |
