ZNF280A Promotes Lung Adenocarcinoma Development through Regulating the Expression of EIF3C

Background: Lung cancer is the most commonly diagnosed malignant tumor worldwide. Lung adenocarcinoma (LUAD) is the most common histological subtype in non-small cell lung cancer (NSCLC). The relationship between ZNF280A and LUAD has not been demonstrated and remains unclear. Methods: In this study, it was demonstrated that ZNF280A was upregulated in LUAD tissues compared with the normal tissues. Further investigations indicated that the overexpression/knockdown of ZNF280A could promote/inhibit proliferation, colony formation and migration of LUAD cells, while inhibiting/promoting cell apoptosis. Moreover, knockdown of ZNF280A could also suppress tumorigenicity of LUAD cells in vivo . RNA-sequencing followed by Ingenuity pathway analysis (IPA) was performed for exploring downstream of ZNF280A and identified EIF3C as the potential target. Results: Furthermore, our study revealed that knockdown of EIF3C could inhibit development of LUAD in vitro , and alleviate the ZNF280A overexpression induced promotion of LUAD. Conclusions: In conclusion, our study showed, as the first time, ZNF280A as a tumor promotor for LUAD, whose function was carried out probably through the regulation of EIF3C. the relationship between LUAD and ZNF280A, which is rarely investigated in the development of cancer, was studied. The IHC analysis of clinical specimens clarified the upregulated expression of ZNF280A was observed in tumor tissues of LUAD. Moreover, knockdown of ZNF280A significantly inhibited cell proliferation of LUAD, and promoted cell apoptosis through the upregulation of Caspase3, Fas, HSP60, IGFBP-6, TNF-β, TRAILR-1 and TRAILR-2, and downregulation of Bcl-2, CD40, IGF-II, Livin and Survivin. The promotion effects of ZNF280A knockdown on cell apoptosis could also be attributed to the arrest of cell cycle in G2 phase by ZNF280A knockdown. Besides, we also found that ZNF280A overexpression exhibited conversed effects against ZNF280A knockdown on cell proliferation and colony formation ability, while simultaneously promoting cell migration of LUAD cells. All these results recognized ZNF280A as a tumor promotor in the development and metastasis of LUAD. Furthermore, the role of ZNF280A in LUAD was finally proved by in vivo experiments, which showed significantly restrained tumor growth of LUAD upon ZNF280A

for LUAD patients 8-10 . Zinc finger protein is a type of transcription factor with a special "finger-like" domain, which usually exists in various eukaryotes and possesses the function of regulating and controlling gene expression 11 . The most representative characteristic of zinc finger protein family members is that they can produce a short stereoscopic structure model of polypeptide according to their own folding pattern, and maintain the stability of such molecular structure by combining with zinc ions 11 . It has been revealed that zinc finger protein plays critical role in embryonic development, cell differentiation, signal transduction and, especially, the development and progression of human cancers 12 . For example, ZNF280B was identified as a potential mechanism of p53 suppression in prostate cancer, which promoted the development of prostate cancer. ZNF280A, which encodes a zinc finger protein with C 2 H 2 motif, was found to be potentially involved in mantle cell lymphoma 13 . However, the relationship between ZNF280A and most types of human cancers including LUAD remains unclear.
In this study, relatively high expression of ZNF280A was observed in lung cancer tissues in comparison with normal tissues, which was significantly correlated with more serious disease and poorer prognosis. Loss-of-function and gain-of-function studies revealed the regulatory role of ZNF280A in the development and progression of LUAD by influencing cell proliferation, colony formation, cell apoptosis, cell cycle distribution and cell migration. The xenografts formed by inoculation of cells with ZNF280A knockdown progress much slower relative to the control group.
Furthermore, EIF3C was screened as the potential downstream of ZNF280A to mediate the regulation of LUAD development. In a word, this study identified ZNF280A as an oncogene-like factor in the development of LUAD, which may be used as an effective therapeutic target in LUAD treatment.

Materials And Methods
Cell lines and cell transfection A549 and NCI-H1299 cells were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences and NCI-H1299 were cultured in RPMI-1640 medium (Gibco) with 10% FBS and A549 was maintained in McCoy's 5A Medium with 10% FBS. All cells were cultured in a humidified cell culture incubator at 37 °C under 5% CO 2 with culture medium changed every 72 h.
For stable gene expressing, lipofectamine RNAimax (Cat. #13778075, Thermo fish) were used for cell A549 and NCI-H1299 transfection with lentiviral plasmids collected. Cells were harvested after 72 h culturing, and cell infection efficiency was valued with LV-shCtrl cells as control.

Immunohistochemical (ihc) Staining
Human lung cancer and para-normal tissue chip (Cat. #HLugA180Su05, Shanghai Outdo Biotech Company) was used and patients' information was collected. For IHC staining, deparaffinized and rehydrated tissue sections were blocked and incubated with primary antibody ZNF280A (Cat. #bs-12839R, BIOSS) and followed incubated by secondary antibody. DAB color was developed with diaminobenzene and hematoxylin. Slides were pictured with microscopic and viewed with ImageScope and CaseViewer. All slides were examined randomly by two independent pathologists and IHC outcomes were determined by staining percentage and intensity scores. Staining percentage scores were classified as: 1 (1%-24%), 2 (25%-49%), 3 (50%-74%) and 4 (75%-100%). Staining intensity were scored 0 (Signalless color) to 3 (light yellow, brown and dark brown). Antibodies used in IHC were listed in Table S1.
Rna Interference And Plasmids Packaging shRNA sequences targeting human ZNF280A and EIF3C gene were designed and cDNAs were synthesized by Shanghai Yibeirui Bioscienceres, Co., Ltd. and subsequently cloned into luciferaselabelled BR-V-108 vector. In addition, ZNF280A was amplified and cloned into the BR-V112 vector after double digestion by BamHI and AgeI, and sequenced. Lentiviral particles were collected, following co-transfection using pHelper 1.0 and pHelper 2.0 vector for plasmids packaging. The sequences used were listed in Table S2.

Rna Extraction And Rt-qpcr
After 72 h for ZNF280A and/or EIF3C RNA expressing, A549 and NCI-H1299 cells in triplicate were fully lysed and total RNA was extracted using TRIzol reagent (Sigma). The RNA quality was evaluated by Nanodrop 2000/2000C spectrophotometer (Thermo Fisher Scientific). cDNA was reversely transcribed from RNA using Promega M-MLV Kit (Promega) and qPCR was performed with SYBR Green mastermixs Kit (Vazyme) by applying Biosystems 7500 Sequence Detection system. GAPDH was acted as inner control, and the primers used for the PCR reaction were showed in Table S3. The relative quantitative analysis in gene expression data were analyzed by the 2 −ΔΔCt method.
Western blotting (WB), co-immunoprecipitation (Co-IP) and Human Apoptosis Antibody Array Cells were lysed in ice-cold RIPA buffer (Millipore), and the protein were collected and the concentration was detected by a BCA Protein Assay Kit (HyClone-Pierce). Protein samples (20 µg per lane) were separated by 10% SDS-PAGE (Invitrogen), and transferred onto PVDF membranes at 4 °C.
The membranes were blocked with TBST solution of 5% degreased milk at room temperature for 1 h and incubated with primary antibodies and GAPDH antibodies at 4 °C overnight. Then the membranes were incubated with secondary antibody HRP goat anti-rabbit IgG for 2 h at room temperature. The blots were visualized by enhanced chemiluminescence (ECL) (Amersham).
For Co-IP, prepared proteins were immunoprecipitated by anti-ZNF280A, EIF3C or GAPDH antibody and then subjected to WB with antibody to ZNF280A and EIF3C and related secondary antibodies.
For Human Apoptosis Antibody Array, briefly, 20 µg total proteins were cultured with the antibodycoated array membranes and then continuing incubated with HRP linked Streptavidin conjugate.
All the antibodies used in western blotting were listed in Table S1.

Cell Proliferation Analysis
The cell viability was determined by MTT assay, briefly, transfected A549 and NCI-H1299 cells were stained with MTT reagent (5 mg/mL, GenView) and Formazan was dissolved by DMSO solution. The absorbance values at 490 nm were measured by microplate reader (Tecan) and the reference wavelength was 570 nm.
Cell proliferation rate was analyzed by Celigo cell counting assay. Briefly, targeting cells were seeded at a 96-well plate with 2,000 cells per well. The plate was continuously detected by Celigo (Nexcelom) for 5 days at the same time.
For colony formation assay, cells in the logarithmic growth phase were seeded into 6-well plates in triplicate and further cultured for 8 days. Cell clones were fixed with 4% paraformaldehyde and stained with Giemsa. Then clones were photographed under a fluorescence microscope (Olympus) and colony number (clone contains more than 50 cells) was counted.

Cell Apoptosis And Cells Cycle Assay
The flow cytometric methods of identifying apoptotic cells was applied using Annexin V-APC Apoptosis kit (Cat. #88-8007, eBioscience). For cells cycle assay, cells were stained with 1 mL PI staining solution (40 × PI, 2 mg/mL: 100 × RNase, 10 mg/mL: 1 × PBS = 25:10:1000). FACScan and FlowJo 7.6.1 (Ashland) was used for analyze. Cell apoptosis was measured and the percentage of the cells in G0-G1, S, and G2-M phase were counted and compared.

Cell Migration Assays
In order to analysis the migration ability of transfected cells in our research, wound healing assay and Transwell assay were performed. For wound healing assay, lentivirus transfected A549 and NCI-H1299 cells (5 × 10 4 cells/well) were plated into 96-well plates for culturing. Scratches were made by a 96 wounding replicator (VP scientific). Photographs were taken by a fluorescence microscope at 0 h, 8 h and 24 h and cell locations were recorded, respectively. Cell migration rates of each cell group were calculated. In transwell assay, cells were seeded into a 24-well plate in the upper chambers, and medium supplemented with 30% FBS was added into in the lower chambers. Cells were fixed with 4% formaldehyde and stained by Giemsa and the migration ability of cells was analyzed.

High-throughput Rna Sequencing
Total RNA from NCI-H1299-shCtrl and NCI-H1299-shZNF280A cells was extracted using TRIzol. RNA quantity and quality were assessed with a Thermo Nanodrop 2000 (1.7 < A260/A280 < 2.2, Thermo Fisher Scientific). Affymetrix PrimeView Human Gene Expression Arrays (Thermo Fisher Scientific) were used for microarray analysis to obtain gene expression profiles according to the manufacturer's instructions. Significantly differentially expressed genes were selected based on P < 0.05 and |Fold Change| > 1.3. KEGG pathway enrichment analysis was performed for all significant differentially expressed genes.

Animal Experiments
All animal studies were approved by Ethics committee of Peking Union Medical College Hospital.
Female BALB/c nude mice were purchased from Shanghai Lingchang Experimental Animals Co., Ltd.
For tumorigenicity, 5 × 10 6 lentivirus (shCtrl or shZNF280A) transfected NCI-H1299 cells were subcutaneously injected into each mouse (4-week-old, n = 10 per group). Mice's weight and tumor sizes were recorded 2 times per week and the volume of tumor were calculated as π/6 × L × W 2 (W, width at the widest point; L, perpendicular width). Finally, the tumor burden was assessed by bioluminescence imaging with non-invasive IVIS Spectrum Imaging System (Perkin Elmer). Mice were sacrificed then tumors were extracted and imaged.

Ki67 Immunostaining Assay
Mice tumor sections were fixed in 4% paraformaldehyde. Paraffin embedded 5 µm sections were made for H&E and IHC staining. We added citric acid buffer for antigen retrieval at 120 °C. Sections were blocked using PBS-H 2 O 2 with 0.1% Tween 20. Ki-67 antibody was added for incubating at 4 °C overnight and then secondary antibodies were added as well. DAB color was developed with diaminobenzene and hematoxylin. Stained slides were pictured with a microscopic.

Statistical Analyses
Each experiment was repeated three times and the data was shown as mean ± SD. Categorical variables were expressed as percentages. The significance between groups was determined using the two-tailed Student's t test or one-way ANOVA analysis. Relationship between ZNF280A expression and tumor characteristics in lung cancer patients with was analyzed using Mann-Whitney U analysis and Spearman grade correlation analysis. Statistical significance was calculated by SPSS 22.0 (IBM) and P value < 0.05 was considered statistically significant. Graphs were made using GraphPad Prism 6.01 (Graphpad Software).

ZNF280A is upregulated in LUAD tissues and expressed in LUAD cells
For the sake of exploring the role of ZNF280A in LUAD, IHC analysis was employed to tell the difference in the expression of ZNF280A in LUAD tissues and normal tissues, indicating the upregulated expression of ZNF280A in LUAD (Fig. 1A). The statistical analysis of expression data collected from 92 LUAD tissues and 70 normal tissues also exhibited the generally higher expression of ZNF280A in LUAD (P < 0.001, Table 1). Correlation analysis between ZNF280A expression and clinical characteristics of patients with LUAD revealed that ZNF280A expression was significantly upregulated in patients with more advanced tumor grade, tumor stage and higher risk of lymphatic metastasis (P < 0.05, Fig. 1A and Table 2), which could also be further verified by performing the poorer prognosis of LUAD patients with relatively higher ZNF280A expression (Fig. 1B).
Additionally, based on the detection of endogenous expression of ZNF280A in LUAD cell lines, A549 and NCI-H1299 cells with relatively high ZNF280A expression was selected for constructing ZNF280A knockdown cell model for subsequent investigations (Fig. 1C).  ZNF280A knockdown inhibited LUAD development in vitro ZNF280A deficiency cell model was constructed through the transfection of lentivirus designed for silencing ZNF280A to elucidate its detailed function in LUAD. The fluorescence signal observed in > 80% cells proved the successful infection ( Figure S1), and the significant downregulation of ZNF280A mRNA and protein levels detected by qPCR (P < 0.001) and western blotting ( Fig. 2A), respectively, confirmed the successful knockdown of ZNF280A in both cell lines. The outcomes of MTT assay showed that cells with ZNF280A depletion (shZNF280A) grew much slower than that without ZNF280A depletion (shCtrl) (P < 0.001, Fig. 2B). As another key factor in cell proliferation, cell apoptosis of  Figure S2). Furthermore, we employed wound-healing and Trasnswell assays to potentiate the decreased cell mobility of A549 and NCI-H1299 cells in shZNF280A groups (P < 0.001, Fig. 2F). Altogether, we supposed that ZNF280A may play a vital role in the development of LUAD through regulating cell apoptosis, colony formation, cell apoptosis and cell migration.

ZNF280A knockdown inhibited tumor growth of LUAD in vivo
After successfully constructing and culturing mice model through injection of NCI-H1299 cells with or without ZNF280A knockdown, the results of in vivo bioluminescence imaging showed markedly weaker total bioluminescence intensity, as well as smaller tumor burden, in shZNF280A group (P < 0.001, Fig. 3A-3B). Moreover, the smaller volume and lighter weight of solid tumors in the shZNF280A group also suggested that tumor growth slowed down upon silence of ZNF280A (P < 0.001, Fig. 3C-3E). Consistently, the lower Ki67 index, as well as lower proliferative activity, detected in the tumors removed from mice of shZNF280A groups further explained the above observations (Fig. 3F).
The potential of EIF3C as the downstream of ZNF280A in the regulation of LUAD Given the basically clear regulatory role of ZNF280A in LUAD, we still wondered the underlying mechanism. Therefore, a 3 v 3 RNA-seq was conducted to identify differentially expressed genes  (Fig. 4A-4D). Among them, EIF3C was supposed to be a promising candidate as the target of ZNF280A. Noteworthy, the expression of EIF3C showed a similar pattern with ZNF280A in LUAD tissues: higher expression in LUAD tissues than normal tissues ( Fig. 4E). More specifically, the direct interaction between ZNF280A and EIF3C was clearly indicated by co-IP in NCI-H1299 cells, which was shown in Fig. 4F. In a word, EIF3C was identified as a potential target of ZNF280A during regulating LUAD, which would be further verified by in vitro investigations.

Knockdown of EIF3C blocked development of LUAD in vitro
In order to illuminate the role of EIF3C in LUAD, EIF3C knockdown cell model was constructed and verified using similar method as mentioned above. Among three shRNAs designed for EIF3C knockdown, RNAi-11091 was shown to possess the highest knockdown efficiency and utilized in subsequent experiments (P < 0.001, Figure S4A-S4B). After further verification of EIF3C knockdown by qPCR and western blotting ( Fig. 5A-5B), NCI-H1299 cells with or without EIF3C knockdown were subjected to Celigo cell counting assay which showed the significantly restrained cell proliferation by EIF3C knockdown (P < 0.001, Fig. 5C). Consistently, we also found that knockdown of EIF3C significantly suppressed the colony formation ability of NCI-H1299 cells (P < 0.001, Fig. 5D). Similar with ZNF280A knockdown, an 8.5-fold elevation of cell apoptosis rate by EIF3C knockdown could be observed in NCI-H1299 cells (P < 0.001, Fig. 5E). More importantly, it was demonstrated by woundhealing and Transwell assays that knockdown of EIF3C could significantly inhibit cell migration ability of NCI-H1299 cells (P < 0.001, Fig. 5F and 5G). Therefore, it could be concluded that knockdown of EIF3C exhibited similar inhibition effects on LUAD with ZNF280A knockdown.

Eif3c Knockdown Alleviated Znf280a Overexpression Induced Promotion Of Luad
In order to clarify the synergistic effect of ZNF280A and EIF3C on LUAD, NCI-H1299 cells with ZNF280A overexpression or simultaneous ZNF280A overexpression and EIF3C knockdown were constructed. The effects of ZNF280A overexpression on functions of NCI-H1299 cells were preliminarily investigated following the detection of transfection efficiency (> 80%, Figure S5A) by fluorescent imaging and verification of knockdown efficiency by qPCR and western blotting (P < 0.001, Figure S5B-S5C). It was demonstrated that ZNF280A overexpression significantly promoted proliferation (P < 0.001, Fig. 6A) and colony formation ability (P < 0.001, Fig. 6B) of NCI-H1299 cells, which was in contrast with the results of ZNF280A and EIF3C knockdown. Interestingly, ZNF280A overexpression only exhibited ignorable influence on cell apoptosis of NCI-H1299 cells without statistical significance (Fig. 6C). Furthermore, we also found that ZNF280A overexpression significantly promoted cell migration ability of NCI-H1299 cells in both wound-healing and Transwell assays (P < 0.01, Fig. 6D and 6E). On the other hand, successful transfection, upregulation of ZNF280A and downregulation of EIF3C were also proved in NCI-H1299 of ZNF280A + shEIF3C group (P < 0.05, Figure S6). An overall inhibition effect on cell proliferation was observed with simultaneous ZNF280A overexpression and EIF3C knockdown (P < 0.001, Fig. 6A). Moreover, subsequent experiments showed that the effects of ZNF280A overexpression on colony formation (P < 0.001, Fig. 6B), cell apoptosis (P < 0.05, Fig. 6C) and cell migration (P < 0.01, Fig. 6D for wound-healing assay and 6E for Transwell assay) could be attenuated or even reversed by EIF3C knockdown. In a word, these results suggested that ZNF280A may execute regulatory effects on LUAD through the regulation of EIF3C.

Discussion
Zinc finger proteins are a group of transcription factors with special finger-like domains. They can generate finger-like structures by self-folding and bind to zinc ions to maintain stability 14  as target of miR-195-5p in the multi-drug resistance of gastric cancer 16 . ZNF668 was reported to be capable of suppressing the invasion and migration of non-small cell lung cancer through regulation of EMT related factors 17 . In bladder cancer, a member of zinc finger protein family ZNF224 was found to form complex with DEPDC1, inhibition of which could potentially repress bladder carcinogenesis 18 .
In this study, the relationship between LUAD and ZNF280A, which is rarely investigated in the Eukaryotic initiation factor 3 (EIF3) is a multi-subunit complex, which was first isolated and purified from rabbit reticulocytes 19 . The EIF3 family of mammals is composed of 13 members, EIF3A~EIF3M.
EIF3 subunits are usually located in the cytoplasm, while EIF3A, EIF3E and EIF3K can participate in the regulation of protein translation between cytoplasm and nucleus due to their special structure 20 . In the process of eukaryotic cell translation initiation, EIF3 can bind directly to small 40S ribosome subunits, thus promoting the formation of the eukaryotic initiation factor 2-triphosphate-amino acid-tRNA (Met-tRNA) ternary complex of the 43S subunit precursor complex, and regulating the synthesis of protein in the process of protein translation 21 . Recent studies showed that EIF3 plays an important role in the occurrence and development of malignant tumors 22,23 . For example, EIF3A, the largest subunit of EIF3 family, is highly expressed in various malignant tumor tissues and has been used as a potential target for anti-cancer drugs 24 . As one of the core subunits of EIF3, EIF3C was also demonstrated to be involved in the development and progression of several types of malignant tumors such as ovarian cancer 25,26 , renal cell carcinoma 27 , osteosarcoma 28 and cervical cancer 29 .
However, the association between EIF3C and LUAD is still not clear and rarely reported.
Herein, we found that EIF3C knockdown could significantly inhibit cell proliferation and colony formation of LUAD cells, while promoting cell apoptosis. Moreover, EIF3C knockdown also suppressed cell migration of LUAD cells. More importantly, the investigation of the synergistic effects of ZNF280A and EIF3C on LUAD showed that EIF3C knockdown could alleviate or even reversed the regulation of LUAD by ZNF280A overexpression. All the results showed the role of EIF3C as a tumor promotor in LUAD and a potential target of ZNF280A.
In conclusion, we found the upregulated expression of ZNF280A and EIF3C in tumor tissues of LUAD.
Both ZNF280A and EIF3C could act as tumor promotors in the development and progression of LUAD, through regulation cell proliferation, colony formation, cell apoptosis and cell migration. More importantly, EIF3C knockdown could attenuate ZNF280A overexpression-induced promotion of LUAD.
Therefore, the regulation of LUAD by ZNF280A through EIF3C make it a potential therapeutic target for LUAD treatment.

Ethics approval and consent to participate
All animal studies were approved by Ethics committee of Peking Union Medical College Hospital.

Consent for publication
Not applicable.

Availability of data and materials
Not applicable.     The expression of Ki67 in sections of xenografts was detected by IHC analysis. Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 on cell migration ability of NCI-H1299 cells were evaluated by wound-healing assay (F) and Transwell assay (G). The representative images were selected from at least 3 independent experiments. Data was shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001