ZNF280A promotes lung adenocarcinoma development by regulating the expression of EIF3C

Abstract

Lung adenocarcinoma (LUAD) is the most common histological subtype in non-small cell lung cancer, which is the malignant tumor with the highest mortality and morbidity in the world. Herein, ZNF280A, a member of the zinc finger protein family carrying two consecutive Cys2His2 zinc finger domains, was shown by us to act as a tumor driver in LUAD. The immunohistochemical analysis of ZNF280A in LUAD indicated its positive correlation with tumor grade, pathological stage and lymphatic metastasis, and negative relationship with patients' survival. A loss-of-function study revealed the inhibition of LUAD development by ZNF280A in vitro and in vivo, whereas ZNF280A overexpression induced opposite effects. Statistical analysis of gene expression profiling in LUAD cells with or without ZNF280A knockdown identified EIF3C as a potential downstream of ZNF280A, which possesses similar regulatory effects on phenotypes of LUAD cells with ZNF280A. Moreover, downregulation of EIF3C in ZNF280A-overexpressed cells could attenuate neutralize the ZNF280A-induced promotion of LUAD. In summary, our study demonstrated that ZNF280A may promote the development of LUAD by regulating cell proliferation, apoptosis, cell cycle, and cell migration and probably via interacting EIF3C.

Fig. 1. ZNF280A was upregulated in LUAD tissues and expressed in LUAD cells.

A The expression level of ZNF280A was detected by IHC analysis in LUAD tissues and normal tissues (Scale bar = 50 μm). B The Kaplan–Meier survival analysis showed a significant association between ZNF280A high expression and shorter survival period of LUAD patients. C The mRNA expression of ZNF280A in A549, NCI-H1299, and SPC-A-1 cell lines was detected by qPCR. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. ZNF280A knockdown inhibited LUAD development in vitro.

A, B Cell models with or without ZNF280A knockdown were constructed by infecting shZNF280A or shCtrl. The knockdown efficiency of ZNF280A in A549 and NCI-H1299 cells was assessed by qPCR and western blotting, respectively. B MTT assay was employed to show the effects of ZNF280A on cell proliferation of A549 and NCI-H1299 cells. C Flow cytometry was performed to detect cell apoptosis of A549 and NCI-H1299 cells with or without ZNF280A knockdown. D Cell cycle distribution was estimated in A549 and NCI-H1299 cells with or without ZNF280A knockdown. E, F The effects of ZNF280A on cell migration ability of A549 and NCI-H1299 cells were evaluated by wound-healing assay E and Transwell assay (Scale bar = 100 μm) F. The representative images were selected from at least three independent experiments. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. ZNF280A knockdown inhibited LUAD development in vivo.

A 22 days post injection of NCI-H1299 cells with or without ZNF280A knockdown, the volume of tumors formed in mice was measured and calculated at indicated time intervals. B In vivo imaging was performed to evaluate the tumor burden in mice of shZNF280A and shCtrl groups at day 34 post tumor inoculation. C The bioluminescence intensity was scanned and used as a representation of tumor burden in mice of shZNF280A and shCtrl groups. D, E Mice were killed at day 34 post injection, and the tumors were removed for collecting photos D and weighing E. F The expression of Ki67 in sections of xenografts was detected by IHC analysis (Scale bar = 50 μm). Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. The exploration and verification of downstream underlying ZNF280A-induced regulation of LUAD.

A A PrimeView Human Gene Expression Array was performed to identify the differentially expressed genes (DEGs) between shZNF280A and shCtrl groups of NCI-H1299 cells. B, C qPCR B and western blotting C were used to detect the expression of several selected DEGs in NCI-H1299 cells with or without ZNF280A. D A ZNF280A associated interaction network constructed by IPA analysis revealed the potential linkage between ZNF280A and EIF3C. E The expression of EIF3C in LUAD tissues and normal tissues was evaluated by IHC analysis (Scale bar = 100 μm). Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. EIF3C knockdown inhibited LUAD development in vitro.

A, B Cell models with or without EIF3C knockdown were constructed. The knockdown efficiency of EIF3C in NCI-H1299 cells was assessed by qPCR A and western blotting B, respectively. C Celigo cell counting assay was employed to show the effects of EIF3C on cell proliferation of NCI-H1299 cells. D Colony formation assay was used to evaluate the ability of NCI-H1299 cells with or without EIF3C knockdown to form colonies. E Flow cytometry was performed to detect cell apoptosis of NCI-H1299 cells with or without EIF3C knockdown. F, G The effects of EIF3C on cell migration ability of NCI-H1299 cells were evaluated by wound-healing assay F and Transwell assay (Scale bar = 100 μm) G. The representative images were selected from at least three independent experiments. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6. Knockdown of EIF3C attenuated the effects of LUAD cells by ZNF280A overexpression.

NCI-H1299 cells transfected with Control plasmids, ZNF280A overexpression plasmids, NC(OE + KD), and simultaneous ZNF280A overexpression plasmids and shEIF3C were subjected to the detection of cell proliferation by Celigo cell counting assay (Scale bar = 400 μm) A, colony formation B, cell apoptosis by flow cytometry C, cell migration by wound-healing assay D and cell migration by Transwell assay (Scale bar = 50 μm) E. The representative images were selected from at least three independent experiments. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.