ONCOLOGY LETTERS 15: 4541-4549, 2018 Knockdown of EIF5A2 inhibits the malignant potential of non-small cell lung cancer cells CHENG CHEN1*, BOJIA ZHANG2*, SHANSHAN WU3, YONGXIANG SONG1 and JIAN LI1 1Department of Thoracic Surgery, Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou 563000; 2Department of Infectious Disease, Shanghai 10th People's Hospital, Tongji University School of Medicine, Shanghai 200072; 3Department of Nursing, Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou 563000, P.R. China Received March 16, 2016; Accepted February 23, 2017 DOI: 10.3892/ol.2018.7832 Abstract. Eukaryotic translation initiation factor 5A2 through these proteins. EIF5A2 may therefore serve as a novel (EIF5A2) has been demonstrated to be upregulated in therapeutic target for the treatment of NSCLC. numerous types of human cancer and is associated with cancer progression. However, the expression and role of Introduction EIF5A2 in non-small cell lung cancer (NSCLC) remains unclear. In the present study, the role of EIF5A2 in NSCLC Non-small cell lung cancer (NSCLC) accounts for 85% of was investigated, in addition to the underlying molecular pulmonary malignancies (1) and is one of the leading causes of mechanisms by which EIF5A2 acts. Relative EIF5A2 expres- cancer-associated mortality worldwide. Recurrent and meta- sion levels were determined in NSCLC cells and compared static disease is the main cause of mortality, and the 5-year with levels in non-cancerous lung tissues. Short interfering survival rate for lung cancer is 15% (2). Therefore, in order to (si)RNA targeted against EIF5A2 was used to knock down improve the prognosis for patients with NSCLC, it is impor- EIF5A2 levels in NSCLC cells. Cell proliferation, apoptosis tant to understand the underlying molecular mechanisms that rate, migration ability and invasion ability were determined contribute to tumor recurrence and metastases, and to identify in untreated and siRNA-treated NSCLC cells, in addition to the relevant markers and targets for treatments. the relative protein expression levels of various tumorigenic The epithelial-mesenchymal transition (EMT) is a complex proteins and E-cadherin. EIF5A2 expression was signifi- but reversible process whereby epithelial cells acquire a mesen- cantly higher in NSCLC tissues compared with adjacent chymal phenotype (3). EMT leads to the loss of the epithelial normal tissues. Knockdown of EIF5A2 in the NSCLC cell marker E-cadherin, which is a cell-cell junction protein. cells significantly inhibited cell proliferation and induced Loss of E-cadherin is associated with NSCLC progression apoptosis. Furthermore, EIF5A2 silencing suppressed cell and metastasis, and with a poor prognosis for patients with migratory and invasive capacities in vitro. Silencing of NSCLC (4). Therefore, determining an effective method to EIF5A2 in the NSCLC cells resulted in the downregulation inhibit EMT in NSCLC may significantly improve the treat- of the tumorigenic proteins, apoptosis regulator Bcl-2 and ment of NSCLC. myc proto-oncogene protein, and upregulation of E-cadherin, Eukaryotic translation initiation factor 5A (EIF5A) is a suggesting that EIF5A2 promotes proliferation and metastasis protein involved in numerous intracellular processes. EIF5A has been demonstrated to serve a role in translation initia- tion, translation elongation, transcription, mRNA turnover and nucleocytoplasmic transport (5). EIF5A arises in two forms (6,7); EIF5A1 serves an essential role in non-malignant cells, although it has also been implicated in cancer (8). EIF5A2 Correspondence to: Dr Jian Li or Dr Yongxiang Song, Department is a purported oncogene that is less widely expressed in normal of Thoracic Surgery, Affiliated Hospital of Zunyi Medical College, tissue (5,9,10); however, EIF5A2 expression has been associ- 201 Dalian Road, Zunyi, Guizhou 563000, P.R. China E‑mail: [email protected] ated with a number of cancer types, including lung cancer. E‑mail: [email protected] Downregulation of EIF5A2 prevents EMT in NSCLC (11), while overexpression of EIF5A2 is an adverse prognostic *Contributed equally marker of survival for patients with stage I NSCLC (12). In addition, it has been demonstrated that inhibition of EIF5A2 Key words: eukaryotic translation initiation factor 5A2, non-small enhances NSCLC sensitivity to chemotherapeutics, prevents cell lung cancer, epithelial-mesenchymal transition, knockdown or reverses EMT, and reduces the migration and invasion capabilities of NSCLC cells (13). The role of EIF5A2 as an oncogene has been well estab- lished in a number of types of cancer, including NSCLC. 4542 CHEN et al: KNOCKDOWN OF EIF5A2 INHIBITS THE MALIGNANT POTENTIAL OF NSCLC CELLS However, its role in NSCLC cells is not clear. In the present supplemented with 100 U/ml penicillin, 100 mg/ml strepto- study, the role of EIF5A2 in NSCLC was investigated. mycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 10% fetal calf serum (Gibco, Thermo Fisher Scientific, Materials and methods Inc.). Cells were maintained at 37˚C with 5% CO2 in a humidified incubator. Patients and specimens. A total of 47 paired tumor tissue Knockdown of EIF5A2 gene expression in NSCLC cells samples and adjacent normal tissues were obtained from was accomplished using small interfering (si)RNAs targeted primary lung cancer patients undergoing surgical treatment against EIF5A2, which were designed and synthesized by between May 2008 and December 2010 at the Affiliated Shanghai GenePharma Co., Ltd. (Shanghai, China). The Hospital of Zunyi Medical College (Zunyi, China). Among the sequence of EIF5A2 siRNA was as follows: 5'-GGA UCU 47 patients with NSCLC, there were 25 males and 22 females UAA ACU GCC AGA ATT-3', 5'-UUC UGG CAG UUU AAG aged between 47 and 71 years old, with a mean age of 60 years AUC CTT-3'. EIF5A2 siRNA (siRNA1, 5 µmol/ml; siRNA2, old. None of the patients had received adjuvant therapy prior to 10 µmol/ml; siRNA3, 15 µmol/ml) or negative control siRNA surgery. Following removal, the tissue specimens were imme- (5'-UUC UCC GAA CGU GUC ACG UTT-3'; 5'-ACG UGA CAC diately fixed in 4% formaldehyde and embedded in paraffin for GUU CGG AGA ATT-3') was transfected into the H1299 and immunohistochemical staining. All the tissues were processed H460 cells using Lipofectamine® 2000 (Invitrogen; Thermo within 15 min of removal. Each sample was frozen and stored Fisher Scientific, Inc.) according to the manufacturer's at ‑80˚C. The tumor tissue samples were confirmed by patho- protocol. The transfection medium was replaced with culture logical diagnosis, while paired non-cancerous adjacent tisssues medium 6 h after transfection. All subsequent experiments were dissected at least 2 cm away from the tumor border were performed 24 h after transfection and repeated three and were confirmed to lack tumor cells by microscopy. The times. patient clinicopathological features are presented in Table I. The present study was approved by the Ethics Committee of Cell proliferation assay. The cell proliferation assay was the Affiliated Hospital of Zunyi Medical College, and written performed using Cell Counting Kit-8 (CCK-8; Dojindo informed consent was obtained from each patient enrolled in Molecular Technologies, Inc., Kumamoto, Japan), according the study. to the manufacturer's protocol. The H1299 and H460 cells treated with EIF5A2 siRNA or control siRNA were seeded Immunohistochemical staining of EIF5A2 expression. on a 96-well microplate at a density of 5x103 cells/well. Cell Immunohistochemical staining of EIF5A2 was performed to proliferation was assessed at 24, 48 and 72 h. CCK-8 solution assess relative EIF5A2 expression in NSCLC tissue compared (10 µl) was added to each well, and the cells were incubated with adjacent normal tissue, according to a standard immunoper- at 37˚C for an additional 3 h. Optical density (OD) was oxidase staining protocol. EIF5A2 immunohistochemistry was determined at a wavelength of 450 nm using an MRX TC II performed on 4‑µm thick, formalin‑fixed, paraffin‑embedded microplate reader (Dynex Technologies, Chantilly, VA, USA). tissue sections. A rabbit polyclonal antibody anti-EIF5A2 (1:100; cat. no. GTX110510; GeneTex Inc., Irvine, CA, USA) was Cell migration and invasion assays. For the migration used for staining (14) and incubated at 37˚C for 2 h, followed assay, H1299 and H460 cells (2x105) were resuspended in by incubation 1 h with horseradish peroxidase-conjugated goat 100 µl serum-free RPMI medium and loaded into the upper anti-rabbit IgG (1:500; cat. no. ab7090; Abcam, Cambridge, chamber of a modified Boyden chamber (Neuro Probe, Inc., MA, USA), then 3,3'‑diaminobenzidine (DAB) staining solu- Gaithersburg, MD, USA). The lower chamber was filled tion (1:25, cat. no. 070004-D; Beijing CellChip Biotechnology with RPMI medium and vascular endothelial growth factor Co., Ltd., Beijing, China) for 10 min at room temperature. (50 ng/ml). The chamber was incubated at 37˚C for 24 h. The The immunohistochemical staining was evaluated using a lower side of the filter was washed with PBS and fixed with 4% semi-quantitative score method as follows: 0, 0%; 1, <5%; 2, paraformaldehyde. The cells were stained with hematoxylin ~5-50%; and 3, >50% stained cells. In addition, the staining and counted in three random high‑power fields in each well intensity was scored as 0 (‑), 1 (+), 2 (++) or 3 (+++). The final under an Olympus inverted microscope (IX70). score was defined as the sum of the two parameters, and the For the invasion assay, the cells (2x105) were resuspended in samples were grouped as negative (0), weak (1-2), moderate 100 µl of serum-free RPMI medium and placed into the upper (3) and strong (4-6) staining. Negative and weak staining were chamber, which contained an 8-µm microporous membrane defined as low expression of EIF5A2, and moderate and strong insert (Costar; Corning Inc., Corning, NY, USA) coated with staining were defined as high expression of EIF5A2. Only the Matrigel® (BD Biosciences, Franklin Lakes, NJ, USA). The final immunoreaction scores of the moderate and strong groups lower chamber contained 10% FBS plus RPMI-1640 medium.