EIF3B Correlates with Advanced Disease Stages and Poor Prognosis, and It Promotes Proliferation and Inhibits Apoptosis in Non-Small Cell Lung Cancer

Cancer Biomarkers 23 (2018) 291–300 291 DOI 10.3233/CBM-181628 IOS Press EIF3B correlates with advanced disease stages and poor prognosis, and it promotes proliferation and inhibits apoptosis in non-small cell lung cancer Yuan Tiana;1, Ke Zhaob;1, Luer Yuanc, Jialing Lic, Shuangbing Fengc, Yufeng Fengc, Zhongqi Fangd, Hui Lic and Ruoyu Dengc;∗ aDepartment of Medical Examination, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China bDepartment of Thoracic Surgery, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China cShanghai Qeejen Bio-tech Institution, Shanghai, China dAnimal Science and Agricultural College, Yanbian University, Yanji, Jilin, China Abstract. BACKGROUND: Although up-regulation of EIF3B correlates with poor prognosis in carcinomas, the role of EIF3B in non- small cell lung cancer (NSCLC) is rarely known. OBJECTIVE: We aimed to investigate correlation of EIF3B with clinicopathological features and prognosis in NSCLC patients, and clarify its effect on cells proliferation and apoptosis. METHODS: Two hundred and eleven NSCLC patients underwent surgery were retrospectively reviewed. Tumor tissue and paired adjacent tissue were obtained. EIF3B expression was detected by immunohistochemistry, qPCR and western blot. EIF3B inhibitor, blank inhibitor, blank mimic and EIF3B mimic plasmids were transfected to A-549 cells. Cells proliferation and apoptosis were measured by CCK-8 and AV/PI. All processes were repeated for validation in PC9 cells. RESULTS: EIF3B expression increased in tumor tissue compared to paired adjacent tissue, and positively correlated with tumor size, lymph node metastasis and TNM stage. K-M curves revealed patients with EIF3B high expression had shorter DFS and OS, and its high expression independently predicted unfavorable DFS and OS. In vitro, EIF3B expression increased in NSCLC cells compared to normal cells. EIF3B increased cells proliferation but inhibited cells apoptosis. CONCLUSIONS: EIF3B overexpression correlates with advanced disease conditions and poor prognosis, and it promotes cells proliferation while inhibits apoptosis in NSCLC. Keywords: EIF3B, NSCLC, clinicopathological features, prognosis, proliferation, apoptosis 1. Introduction Non-small cell lung cancer (NSCLC), accounting for over 85% of lung cancer, is one of the most fre- 1Yuan Tian and Ke Zhao contributed equally to this work. quently diagnosed malignancies and the leading cause ∗ Corresponding author: Ruoyu Deng, Shanghai Qeejen Bio-tech of cancer related death worldwide [1–3]. Due to lack Institution, 2500 Siping Road, Shanghai 200433, China. Tel.: +86 13761145675; Fax: +86 021 35080019; E-mail: jessdeng@qeejen. of obvious clinical symptoms, most patients are diag- com. nosed as NSCLC at an advanced disease stage, con- ISSN 1574-0153/18/$35.00 c 2018 – IOS Press and the authors. All rights reserved 292 Y. Tian et al. / EIF3B in non-small cell lung cancer tributing to worse prognosis, or even death [2,4,5]. 2.3. Data collection, follow up and survival Although imaging technology, targeted therapies, im- calculation munotherapies and individual treatment strategies have been greatly improved, the prognosis of NSCLC is Baseline information including age, gender, patho- still not satisfied since the 5-year survival rate is just logical grade, tumor size, lymph node metastasis and nearly 15% [6]. Therefore, exploring new and convinc- TNM stage were reviewed and collected by Electronic ing biomarkers for prognosis and clarifying novel un- Medical Record System of The Central Hospital of derlying mechanisms of tumer progression of NSCLC Wuhan in this study. TNM stage was classified accord- are necessary. ing to 7th edition of the American Joint Committee on Eukaryotic initiation factors (eIFs), a series of pro- Cancer (ATCC) cancer staging manual. The neoplastic teins or protein complexes, are essential in the rate- grading is a measure of cell anaplasia (reversion of dif- limiting initiation phase of eukaryotic translation, ferentiation) in the sampled tumor and is based on the which play critical roles in the progress and progno- resemblance of the tumor to the tissue of origin. Patho- sis of several carcinomas [7–10]. As a member of the logical grade are classified as 1, 2, 3, or 4 depending eIFs family, Eukaryotic translation initiation factor 3 subunit B (EIF3B) is highly expressed in several car- on the amount of abnormality, among which grade G1 cinomas, including bladder, breast, and prostate can- meant well differentiated (low grade), G2 meant mod- cers [10–12]. Despite of the fact that up-regulation erately differentiated (high grade), G3 meant poorly of EIF3B is correlated with poor prognosis in many differentiated (high grade) and G4 meant undifferenti- carcinomas, the role of EIF3B in NSCLC is rarely ated (high grade) [15]. Patients were followed up reg- known [11,13,14]. Therefore, our purpose was to in- ularly and the median follow duration was 49 months, vestigate the correlation of EIF3B expression with last follow up date was 2017/12/31. Disease-free sur- clinicopathological features and prognosis in NSCLC vival (DFS) was calculated from the time of surgery to patients, as well as clarify the effect of EIF3B on the the date of documented local or distant recurrence, or cells proliferation and apoptosis in NSCLC cells. death from any cause. overall survival (OS) was calculated from the time of surgery to the date of death from any cause. 2. Methods 2.4. Immunohistochemistry (IHC) assay for EIF3B 2.1. Participants expression in tissues Two hundred and eleven NSCLC patients underwent surgery at The Central Hospital of Wuhan from Formaldehyde fixed, paraffin embedded tumor tis- Jan 2009 to Dec 2012 were retrospectively reviewed sue sample and paired adjacent tissue sample were in this study. The inclusion criteria were as followed: obtained from the Specimen Repository of The Cen- (1) Diagnosed as primary NSCLC based on clinical tral Hospital of Wuhan. After being deparaffinized and and pathological confirmation. (2) Age above 18 years. rehydrated, antigen retrieval was conducted in tissue (3) Completed information of tumor features was sections in Tris-Ethylene Diamine Tetraacetic Acid available to retrieve. (4) Regularly follow-up patients. (EDTA). Then endogenous peroxidase was block by (5) Formaldehyde fixed, paraffin embedded tumor tis- H2O2, and sections were immersed with 4% bovine sue sample and paired adjacent tissue sample were serum albumin for blocking nonspecific endogenous available to obtain. The exclusion criteria were as fol- antigens. Subsequently, sections were incubated with lows: (1) Received neoadjuvant therapy before surgery. anti-EIF3B antibody (Abcam, USA) as primary an- (2) History of other solid tumors or hematologic ma- tibody at 4◦C overnight. After washing, the sections lignancy. were incubated with horseradish peroxidase (HRP)- conjugated anti-IgG (Abcam, USA) as secondary an- 2.2. Ethics approval tibody at room temperature for 30 minutes. After- This present study was approved by the Ethics Com- wards, tissue sections were washed and treated with mittee of The Central Hospital of Wuhan, and was con- diaminobenzidine (DAB), followed by counterstaining ducted under the Statement of Helsinki. All the par- with hematoxylin. Then, sections were dehydrated and ticipants or their guardians signed the informed con- mounted. sents or orally agreed with the informed consents by As to the expression of EIF3B assessment, the posi- telephone with tape recording. tive cells were observed and counted under a light mi- Y. Tian et al. / EIF3B in non-small cell lung cancer 293 croscopy by two specialists without knowing any in- 2.7. EIF3B plasmids transfection and subsequent formation about patients. In each slide, 100 cells in 5 assays in A-549 cells high-power fields (HPF, × 400) were counted to eval- uate the intensity of positive cells. Staining intensity EIF3B inhibitor plasmid, blank inhibitor plasmid, was scored as 0 (negative), 1 (weak), 2 (moderate), to 3 blank mimic plasmid and EIF3B mimic plasmid were (strong), while the grading scale for labeling frequency designed and constructed by a Shanghai Gemma com- ranged from 0 (0%), 1 (1%–25%), 2 (26%–50%), 3 pany (Shanghai, China). These plasmids were trans- (51%–75%), and 4 (76%–100%) on the basis of the fected into A-549 cells as EIF3B (-), NC (-), NC (+) percentage of positively stained cells. Finally, Multi- and EIF3B (+) groups. qPCR assay and Western Blot plying the score of staining intensity by the labeling assay were performed to detect the mRNA and protein frequency score was used to divide sections into two expressions of EIF3B at 24 h respectively. counting kit- groups: EIF3B low expression group with final score 8 (CCK-8) (Abcam, USA) assay was performed to de- 6 3; and EIF3B high expression group with final score termine cells proliferation at 0 h, 24 h, 48 h and 72 h. > 3. Annexin V (AV) apoptosis detection kit with propid- ium iodide (PI) (Invitrogen, USA) was used to detect cells apoptosis rate at 72 h. 2.5. Cells culture 2.8. Validation in PC9 cells NSCLC cell lines including A-549, NCI-H1650, NCI-H1299 and PC9 as well as normal lung epithe- In order to further validate the effect of EIF3B on lial cells (DEAS-2B) were purchased from Shanghai cells proliferation and apoptosis in NSCLC, additional Institutes for Biological Science (Shanghai, China) or experiments in PC9 cells were carried out. EIF3B in- obtained from Shanghai QeeJen Bio-tech Institution hibitor plasmid, blank inhibitor plasmid, blank mimic (Shanghai, China). A-549 cells were cultured in RPMI plasmid and EIF3B mimic plasmid were transfected F12K medium (Gibco, USA) supplemented with 10% into PC5 cells, which were classified as EIF3B (-), NC (v/v) fetal bovine serum (FBS) (Gibco, USA) and an- (-), NC (+) and EIF3B (+) groups respectively. After tibiotics (100 units/ml penicillin and 100 µg/ml strep- transfection at 0 h, 24 h, 48 h and 72 h, CCK-8 assay tomycin) (Corning, USA). NCI-H1650 cells and NCI- (Abcam, USA) for evaluation of cells proliferation was H1299 cells were cultured in RPMI-1640 medium carried out.

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