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

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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. At transfection at 72 h, AV-PI (Invitrogen, (Gibco, USA) supplemented with 10% (v/v) FBS and USA) for cells apoptosis rate was performed. antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin). PC9 cells were cultured in Dulbecco’s 2.9. qPCR assay modified Eagles medium (DMEM) (Gibco, USA) supplemented with 10% (FBS), 100 units/ml penicillin The mRNA level of EIF3B was detected by qPCR and 100 µg/ml streptomycin. DEAS-2B cells were cul- as follows: According to the manufacturer’s instruc- tured in MEM medium (Gibco, USA) supplemented tions, total RNA samples were extracted with TRI- with 10% (FBS), 100 units/ml penicillin and 100 µg/ml zol reagent (Invitrogen, USA). Total RNA was quanti- streptomycin. All cells were incubated at 37◦C with fied by OD 260 (Takara, Japan). Then, 1 µg of RNA 5% CO2. per group was applied to cDNA synthesis separately. Following, those cDNA were subjected to qPCR by SYBR Green kit (Takara, Japan). The PCR profile was 2.6. Detection of EIF3B expression in NSCLC cell 95◦C for 5 min, and 40 cycles of 95◦C for 5 s, 61◦C lines for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal reference. EIF3B Total RNA was extracted from NSCLC cell lines (A- expression was calculated by the 2−∆∆ct method. 549, NCI-H1650, NCI-H1299 and PC9) and normal Primer sequences used in this study were as follows: lung epithelial cells (DEAS-2B) by TRIzol Reagents EIF3B Forward primer: 5’-CGGTGCCTTAGCGTTT (Invitrogen, USA). Quantitative polymerase chain re- GTG-3’; EIF3B Re-verse primer: 5’-CGGTCCTTGT action (qPCR) assay and Western Blot assay were per- TGTTCTTCTGC-3’; GAPDH Forward primer: 5’-TG formed to detect the mRNA and protein expressions of ACTTCAACAGCGACACCCA-3’; GAPDH Reverse EIF3B respectively. pri-mer: 5’-CACCCTGTTGCTGTAGCCAAA-3’. 294 Y. Tian et al. / EIF3B in non-small cell lung cancer

2.10. Western blot assay Table 1 Characteristics of NSCLC patients Cells were lysed with radioimmunoprecipitation ly- Items NSCLC patients (N = 211) sis buffer (1% protease inhibitor cocktail, 1% phenyl- Age (years) 61.31 ± 10.35 Gender (male/female) 143/68 methanesulfonyl fluoride), and clarified by centrifu- Pathological grade gation. Subsequently, the protein concentration was G1 (n/%) 30 (14.2) detected by the BCATM protein assay kit (Pierce, G2 (n/%) 134 (63.5) USA). Proteins were then separated on sodium dodecyl G3 (n/%) 47 (22.3) Tumor size sulfate-polyacrylamide (SDS) gel electrophoresis and 6 5 cm (n/%) 122 (57.8) transferred to PVDF membrane. After blocked with > 5 cm (n/%) 89 (42.2) 5% non-fat dried milk in PBST at room temperature Lymph node metastasis for 1 h, PVDF membrane was incubated with EIF3B or Negative (n/%) 133 (63.0) Positive (n/%) 78 (37.0) GAPDH antibodies, and finally incubated with HRP- TNM stage conjugated secondary antibody (Cell Signaling Tech- I 71 (33.6) nology, USA). The specific band was visualized by an II 72 (34.1) ECL advanced Western blot analysis detection kit (BD, III 68 (32.2) USA). Data were presented as mean ± standard deviation or count (percentage). NSCLC, non-small-cell lung cancer.

2.11. CCK-8 assay Table 2 Comparison of EIF3B expression between tumor tissue and paired Ten µl CCK-8 and 90 µl medium were added into adjacent tissue A-549 cells, and the A-549 cells were then incubated Items EIF3B expression P value ◦ under 5% CO2 at 37 C. Optical density (OD) value Low High was determined by microplate reader (Biotech, USA) expression expression and represented the cells proliferation ability. Tumor tissue 101 (47.9) 110 (52.1) < 0.001 Paired adjacent tissue 162 (76.8) 49 (23.2) 2.12. AV/PI assay Data were presented as count (percentage). Comparison was determined by McNemar test. P < 0.05 was considered significant. EIF3B, eukaryotic translation initiation factors 3B. After being digested by pancreatin and washed by phosphate buffer solution (PBS), cells were suspended 3. Results in 100 µl Blinding Buffer. Subsequently, 2 µl AV (In- vitrogen, USA) was subsequently added into the cells 3.1. Baseline characteristics and the cells were placed in the darkness for 15 min on the ice. Subsequently, 1 µl PI (Invitrogen, USA) As shown in Table1, the mean age of 211 NSCLC was added just before flow cytometry (FCM) assay and patients was 61.31 ± 10.35 years. There were 143 the results were analyzed by FCM (Becton Dickinson, males and 68 females. According to the pathological USA). grade, the numbers of patients in G1 grade, G2 grade, and G3 grade were 30 (14.2%), 134 (63.5%), and 47 2.13. Statistics (22.3%) respectively. There were 122 (57.8%) patients with tumor size 6 5 cm, while 89 (42.2%) patients Statistical analysis was performed using SPSS Soft- with tumor size > 5 cm. 133 (63.0%) patients pre- ware 21.0 (IBM, USA) and GraphPad Prism 5.01 sented with negative lymph node metastasis, while 78 (GraphPad, USA). Data were mainly presented as (37.0%) patients presented with positive lymph node mean ± standard deviation or count (percentage). metastasis. As for the TNM stage, the numbers of pa- Comparison between two groups was determined by tients in stage I, stage II and stage III were 71 (33.6%), t test, Chi-square test or McNemar test. DFS and OS 72 (34.1%) and 68 (32.2%) respectively. were analyzed by Kaplan-Meier (K-M) curves and log- rank test. Univariate and multivariate Cox’s propor- 3.2. EIF3B expression in tumor tissue and paired tional hazards regression analysis was performed to de- adjacent tissue termine the baseline factors affecting DFS and OS. The predicting values of baseline factors for DFS and OS. EIF3B expression was detected by IHC assay, which P < 0.05 was considered as significant. disclosed that EIF3B expression was higher in tumor Y. Tian et al. / EIF3B in non-small cell lung cancer 295

Fig. 1. EIF3B expression in tumor tissue and paired adjacent tissue. A: Example of EIF3B high expression in tumor tissue. B: Example of EIF3B low expression in paired adjacent tissue. EIF3B, eukaryotic translation initiation factor 3 subunit B.

Table 3 der (P = 0.189), or pathological grade (P = 0.252) in Correlation of EIF3B expression with clinicopathological features of NSCLC patients NSCLC patients was observed (Table3). Items EIF3B Low EIF3B High P value 3.4. Correlations of EIF3B expression with DFS and expression expression (n = 101) (n = 110) OS in NSCLC patients Age 0.168 6 60 years 49 (53.5) 43 (46.7) K-M curves revealed that NSCLC patients with > 60 years 52 (43.7) 67 (56.3) EIF3B high expression presented with shorter DFS Gender 0.189 (P < 0.001, Fig.2A) and OS ( P < 0.001, Fig.2B) Male 64 (44.8) 79 (55.2) than that in patients with EIF3B low expression. Female 37 (54.4) 31 (45.6) Pathological grade 0.252 G1 18 (60.0) 12 (40.0) 3.5. Factors affecting DFS in NSCLC patients by G2 59 (44.0) 75 (56.0) Cox’s analysis G3 24 (51.1) 23 (48.9) Tumor size < 0.001 Univariate Cox’s proportional hazards regression 6 5 cm 44 (60.7) 48 (39.3) was employed to assess factors inﬂuencing DFS in > 5 cm 27 (30.3) 62 (69.7) Lymph node metastasis < 0.001 NSCLC patients (Table4), which disclosed that EIF3B Negative 79 (59.4) 54 (40.6) high expression (P < 0.001), poor pathological grade Positive 22 (28.2) 56 (71.8) (P < 0.001), and higher TNM stage (P = 0.024) were TNM stage 0.002 associated with shorter DFS. Multivariate Cox’s pro- I 45 (63.4) 26 (36.6) II 33 (45.8) 29 (54.2) portional hazards regression disclosed that EIF3B high III 23 (33.8) 45 (66.2) expression (P < 0.001), age > 60 years (P = 0.044) Data were presented as count (percentage). Comparison was deter- and poor pathological grade (P < 0.001) could inde- mined by Chi-square test. P < 0.05 was considered signiﬁcant. pendently predict shorter DFS in NSCLC patients (Ta- EIF3B, eukaryotic translation initiation factors 3B; NSCLC, non- ble4). small-cell lung cancer. 3.6. Factors affecting OS in NSCLC patients by Cox’s tissue (Fig.1A) compared to paired adjacent tissue analysis (Fig.1B) ( P < 0.001, Table2). As shown in Table5, EIF3B high expression ( P < 3.3. Correlations of EIF3B expression with 0.001), poor pathological grade (P < 0.001) and clinicopathological features in NSCLC patients higher TNM stage (P = 0.003) were correlated with poor OS. Multivariate Cox’s proportional hazards re- EIF3B expression was positively correlated with tu- gression revealed that EIF3B high expression (P < mor size (P < 0.001), lymph node metastasis (P < 0.001) and poor pathological grade (P < 0.001) inde- 0.001) and TNM stage (P = 0.002), while no correla- pendently predict unfavorable OS in NSCLC patients tion of EIF3B expression with age (P = 0.168), gen- (Table5). 296 Y. Tian et al. / EIF3B in non-small cell lung cancer

Table 4 Cox’s analysis of factors affecting DFS Items Univariate Cox’s proportional hazard regression Multivariate Cox’s proportional hazard regression P value HR 95% CI P value HR 95% CI Lower Higher Lower Higher EIF3B high expression < 0.001 1.889 1.408 2.534 < 0.001 1.816 1.337 2.467 Age > 60 years 0.167 1.230 0.917 1.650 0.044 1.368 1.008 1.857 Gender (male) 0.321 1.174 0.860 1.604 0.751 0.949 0.689 1.309 Pathological grade < 0.001 1.506 1.196 1.895 < 0.001 1.577 1.223 2.033 TNM stage 0.024 1.222 1.026 1.454 0.453 1.074 0.892 1.293 Data were presented as P value, HR and 95%CI. Baseline factors affecting DFS were determined by univariate and multivariate Cox’s proportional hazards regression model analysis. P < 0.05 was considered signiﬁcant. Pathological grade was scored as 1-G1, 2-G2, 3-G3. TNM stage was scored as 1-I, 2-II, 3-III. DFS, disease free survival; HR, hazard ratio; CI, conﬁdence interval; EIF3B, eukaryotic translation initiation factors 3B.

Fig. 2. Correlation of EIF3B expression with DFS and OS in NSCLC patients. A: Shorter DFS was observed in EIF3B high expression patients. B: Worse OS was presented in patients with EIF3B high expression. Kaplan-Meier curves and log-rank test were performed for comparison of DFS and OS. P < 0.05 was considered as signiﬁcant. EIF3B, eukaryotic translation initiation factor 3 subunit B. DFS, disease free survival. OS, overall survival. NSCLC, non-small cell lung cancer.

Fig. 3. EIF3B expression in NSCLC cell lines. A: mRNA level of elF3B was increased in NSCLC cell lines (including A-549, NCI-H1299, NCI-H1650 and PC9 cells) compared to normal lung epithelial cell line DEAS-2B, and the most significant elevation was in A-549 cells. B: Protein level of elF3B was upregulated in NSCLC cell lines including A-549, NCI-H1299, NCI-H1650 and PC9 cells compared to normal lung epithelial cell line DEAS-2B, and most significantly increased in A549 cells. Comparison between two groups was determined by t test. P < 0.05 was considered as significant. ∗P < 0.05, ∗∗P < 0.01. EIF3B, eukaryotic translation initiation factor 3 subunit B. NSCLC, non-small cell lung cancer.

3.7. EIF3B expression in NSCLC cell lines sions (Fig.3B) of EIF3B were increased in NSCLC cell lines including A-549, NCI-H1299, NCI-H1650 In order to investigate the effect of ElF3B on NSCLC cells, in vitro experiments were further per- and PC9, and was most signiﬁcantly elevated in A-549 formed. Compared to normal lung epithelial cells cells. Therefore, A-549 cell line was chosen for the fol- DEAS-2B, both mRNA (Fig.3A) and protein expres- lowing in vitro experiments. Y. Tian et al. / EIF3B in non-small cell lung cancer 297

Table 5 Cox’s analysis of factors affecting OS Items Univariate Cox’s proportional hazard regression Multivariate Cox’s proportional hazard regression P value HR 95% CI P value HR 95% CI Lower Higher Lower Higher EIF3B high expression < 0.001 2.018 1.454 2.802 < 0.001 1.929 1.373 2.709 Age > 60 years 0.501 1.117 0.809 1.543 0.186 1.258 0.895 1.769 Gender (male) 0.784 0.954 0.681 1.336 0.122 0.761 0.539 1.076 Pathological grade < 0.001 1.626 1.258 2.102 < 0.001 1.674 1.253 2.236 TNM stage 0.003 1.351 1.110 1.644 0.153 1.167 0.944 1.441 Data were presented as P value, HR and 95% CI. Baseline factors affecting OS were determined by univariate and multivariate Cox’s proportional hazards regression model analysis. P < 0.05 was considered signiﬁcant. Pathological grade was scored as 1-G1, 2-G2, 3-G3. TNM stage was scored as 1-I, 2-II, 3-III. OS, overall survival; HR, hazard ratio; CI, conﬁdence interval; EIF3B, eukaryotic translation initiation factors 3B.

Fig. 4. EIF3B promoted proliferation and inhibited apoptosis of A-549 cells. A: mRNA level of EIF3B was downregulated in EIF3B (-) group and upregulated in EIF3B (+) group compared to corresponding NC groups. B: Protein level of EIF3B was lower in EIF3B (-) group and higher in EIF3B (+) group than corresponding NC groups. C: EIF3B promoted cells proliferation of A549 cells. D and E: EIF3B inhibited cells apoptosis of A549 cells. Comparison between two groups was determined by t test. P < 0.05 was considered as signiﬁcant. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. NC, negative control. EIF3B, eukaryotic translation initiation factor 3 subunit B. 298 Y. Tian et al. / EIF3B in non-small cell lung cancer

Fig. 5. EIF3B induced cells proliferation and suppressed cells apoptosis of PC9 cells. A: mRNA level of EIF3B was decreased in EIF3B (-) group and increased in EIF3B (+) group compared to corresponding NC groups. B: Protein level of EIF3B was decreased in EIF3B (-) group and increased in EIF3B (+) group than corresponding NC groups. C: EIF3B promoted cells proliferation of PC9 cells. D-E: EIF3B inhibited cells apoptosis of PC9 cells. Comparison between two groups was determined by t test. P < 0.05 was considered as signiﬁcant. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. NC, negative control. EIF3B, eukaryotic translation initiation factor 3 subunit B.

3.8. EIF3B promoted proliferation and inhibited apoptosis rate was increased in EIF3B (-) group com- apoptosis in A-549 cells pared to NC (-) group, while it was decreased in EIF3B (+) group compared to NC (+) group at 72 h after trans- Both mRNA and protein expressions of EIF3B were fection (Fig.4D and E), suggesting that EIF3B inhib- decreased in EIF3B (-) group compared to NC (-) ited apoptosis in A-549 cells. group and increased in EIF3B (+) group compared to NC (+) group, indicating the transfection was suc- 3.9. Validation for the effect of EIF3B on cells cessful (Fig.4A and B). At 72 h, cells proliferation proliferation and cells apoptosis in PC9 cells was promoted in EIF3B (+) group compared to NC (+) group, while it was suppressed in EIF3B (-) group To further verify the efficacy of EIF3B on cells pro- compared to NC (-) group (Fig.4C), suggesting that liferation and apoptosis in NSCLC, additional experi- EIF3B promoted proliferation in A-549 cells. Cells ments were repeated in another NSCLC cells line PC9 Y. Tian et al. / EIF3B in non-small cell lung cancer 299 cells. As shown in Fig.5, mRNA and protein expression is associated with an aggressive tumor phenotype sions of EIF3B were evaluated in EIF3B (+) group in patients with clear cell renal cell carcinoma [14]. compared to NC (+) group and reduced in EIF3B (-) Furthermore, EIF3B expression positively correlates group compared to NC (-) group, suggesting the trans- with tumor grade in patients with bladder and prostate fection was successful (Fig.5A and B). Cells prolifera- cancers [11]. Therefore, these previous studies suggest tion was increased in EIF3B (+) group compared to NC that EIF3B overexpression associates with advanced (+) group and was decreased in EIF3B (-) group com- disease conditions in patients with several cancers. In pared to NC (-) group at 72 h (Fig.5C), indicating that line with those previous data, we found that EIF3B ex- EIF3B promoted proliferation in PC9 cells. For cells pression was higher in tumor tissue than that in adja- apoptosis rate, it was decreased in EIF3B (+) group cent tissue, and it was positively correlated with tumor compared to NC (+) group but increased in EIF3B size, lymph node metastasis and TNM stage in NSCLC (-) group compared to NC (-) group at 72 h after trans- patients. The possible reasons are as follows: EIF3B fection (Fig.5D and E), which indicated that EIF3B has effect on promotion of cells proliferation, facilita- suppressed cells apoptosis in PC9 cells. tion of cells migration or inhibition of cells apoptosis via modulating various genes expressions or pathways activations (including decreasing integrin α5 expres- 4. Discussion sion, inhibiting tumor necrosis factor receptor super- family member 21 expression or activating β-catenin In this study, we observed that: (1) EIF3B expres- signaling pathway), thereby contributing to advanced sion was higher in tumor tissue than that in adjacent disease conditions in NSCLC patients [11,13,17]. tissue, and it was positively associated with tumor size, As to the predictive value of EIF3B on the prog- lymph node metastasis and TNM stage. Moreover, nosis of cancers, some studies have been performed. EIF3B overexpression correlated with worse DFS and For instance, esophageal squamous cell carcinoma pa- OS independently in NSCLC patients. (2) EIF3B pro- tients with high EIF3B expression present with poor moted proliferation and inhibited apoptosis in NSCLC DFS and OS [17]. For clear cell renal cell carci- cells. noma, patients with elF3B overexpression also illu- EIFs families, which participate in the eukaryotic minate shorter OS [14]. Additionally, up-regulation translation via regulating interaction between ribosome of EIF3B correlates with poor disease-specific sur- and mRNA, are propo15sed as new therapeutic tar- vival in patients with bladder cancer [11]. Even though gets for carcinomas due to their functions in pro- those previous studies indicate that EIF3B acts as a moting tumorigenesis [14,16,17]. An interesting ex- novel biomarker for worse prognosis in some cancers, periment indicates that down-regulation of eukary- the effect of EIF3B on the prognosis of NSCLC is otic translation initiation factor 5A2 (EIF5A2) sup- still unclear. In the present study, we observed that presses cells growth in ovarian cancer cell line UACC- EIF3B overexpression independently correlated with 1598 [18]. Also, eukaryotic translation initiation fac- worse DFS and OS in NSCLC patients. The possi- tor 3 subunit D (EIF3D) plays a crucial role in pro- ble reasons were as follows: EIF3B could influence moting cells proliferation via affecting G2/M phase ar- expressions of some proteins or activation of path- rest in NSCLC [19]. Additionally, eukaryotic initiation ways (such as reducing apoptosis inhibitory protein translation factor 2α (EIF2α) phosphorylation reduces BCL2 expression or activating the pro-survival Akt cisplatin-induced apoptosis of NSCLC A-549 cells via pathway) to induce cells proliferation and inhibit cells decreasing p38 expression [20]. Hence, those previous apoptosis, thereby devoting to advanced disease condi- data suggest that EIFs may be oncogenes in carcino- tions and finally leading to poor prognosis in NSCLC mas, including NSCLC. patients [14,21,22]. Moreover, elF3B might promote EIF3B, a common member of EIFs family, has been drug resistance via mediating some pathways, includ- observed to be overexpressed in many cancers including activating the Akt pathway, thereby decreasing ing esophageal squamous cell carcinoma, clear cell re- treatment efficacy and resulting in poor prognosis in nal cell carcinoma, and prostate cancers [11,14,17]. In NSCLC patients [21]. addition, up-regulation of EIF3B is found to be posi- In order to investigate the function of EIF3B in tively correlated with tumor depth, lymph node metas- cancers and clarify the underlying mechanisms, some tasis and TNM stage in patients with esophageal squa- in vitro experiments have been performed. For exam- mous cell carcinoma [17]. Also, EIF3B high expres- ple, eIF3b contributes to osteosarcoma cells prolifera- 300 Y. Tian et al. / EIF3B in non-small cell lung cancer tion by regulating tumor necrosis factor receptor super- [9] J.W. Hershey, The role of eIF3 and its individual subunits in family member 21 expression [13]. Also, depletion of cancer, Biochim Biophys Acta 1849 (2015), 792–800. EIF3B inhibits cells growth, prevents cells migration, [10] R. Spilka, C. Ernst, A.K. Mehta and J. Haybaeck, Eukaryotic translation initiation factors in cancer development and pro- hinders cells cycle, as well as disrupts actin cytoskele- gression, Cancer Lett 340 (2013), 9–21. ton and focal adhesions by decreasing the expression [11] H. Wang, Y. Ru, M. Sanchez-Carbayo, X. Wang, J.S. Kieft of integrin α5 in bladder cancer cells [11]. Addition- and D. Theodorescu, Translation initiation factor eIF3b expression in human cancer and its role in tumor growth and ally, knockdown of EIF3B inhibits cells proliferation lung colonization, Clin Cancer Res 19 (2013), 2850–2860. by cell cycle arrest in U87 cells [23]. Therefore, these [12] L. Lin, T. Holbro, G. Alonso, D. Gerosa and M.M. Burger, previous data indicate that EIF3B promotes tumori- Molecular interaction between human tumor marker protein genesis through facilitating cells proliferation and in- p150, the largest subunit of eIF3, and intermediate filament protein K7, J Cell Biochem 80 (2001), 483–490. hibiting cells apoptosis in several carcinomas, while [13] Y.J. Choi, Y.S. Lee, H.W. Lee, D.M. Shim and S.W. Seo, Si- the function of EIF3B in NSCLC cells is still unclear. lencing of translation initiation factor eIF3b promotes apopto- To clarify the role of EIF3B in cells proliferation and sis in osteosarcoma cells, Bone Joint Res 6 (2017), 186–193. apoptosis of NSCLC, a series of in vitro experiments [14] Y. Zang, X. Zhang, L. Yan, G. Gu, D. Li, Y. Zhang, L. Fang, S. Fu, J. Ren and Z. 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