Fig 2: CCDC34 overexpression abolishes the function of MNX1 knockdown on cell cycle and apoptosis in A549 cells. (A) Cell cycle distribution was assessed using flow cytometry analysis. (B) Protein expression of cyclin B1 and p-Histone H2A.X was detected using western blotting. (C) Cell apoptosis rate was detected using flow cytometry analysis. (D) Protein expression of Bcl-2, Bax, cleaved-caspase3 and cleaved-caspase9 was detected using western blotting. ***P<0.001 vs. sh-NC; #P<0.05, ###P<0.001 vs. sh-MNX1+oe-NC. CCDC34, coiled-coil domain-containing 34; MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector).

Fig 3: MNX1 knockdown represses proliferation, migration and invasion of A549 cells. Following transfection with sh-NC or sh-MNX1-1/sh-MNX1-2 in A549 cells, (A) the mRNA level and (B) protein expression of MNX1 were examined using reverse transcription-quantitative PCR and western blotting, respectively. (C) CCK-8 assay was used to examine cell viability. (D) Cell colony formation assay was conducted to assay formed colonies. (E-G) Wound-healing and Transwell assays were performed to examine cell migration and invasion, respectively. Magnification, ×100. (H) Protein expression of Ki67, PCNA, MMP2 and MMP9 was examined using western blotting. *P<0.05, **P<0.01, ***P<0.001 vs. sh-NC. MNX1, Motor neuron and pancreas homeobox 1; sh, short hairpin RNA; NC, negative control (empty vector); CCK-8, Cell Counting Kit-8; PCNA, proliferating cell nuclear antigen.

Fig 4: MNX1 is upregulated in LUAD. GEPIA database exhibited (A) the MNX1 expression level in tumor tissues of LUAD and (B) the overall survival of LUAD patients with high or low expression of MNX1. Human bronchial epithelial cells BEAS-2B and human LUAD cell lines (PC-9, A549, H1975 and H3255) were used to examine the (C) mRNA level and (D) protein expression of MNX1 using reverse transcription-quantitative PCR and western blotting, respectively. *P<0.05, **P<0.01, ***P<0.001 vs. BEAS-2B. MNX1, Motor neuron and pancreas homeobox 1; LUAD, Lung adenocarcinoma; GEPIA, Gene Expression Profiling Interactive Analysis.

Fig 5: MNX1 binds to CCDC34 promoter and transcriptionally regulates CCDC34 expression. (A) GEPIA database exhibited the CCDC34 expression level in tumor tissues of LUAD. *P<0.05. Human bronchial epithelial cells BEAS-2B and human LUAD cell lines A549 were applied to examine the (B) mRNA level and (C) protein expression of CCDC34 using RT-qPCR and western blotting, respectively. ***P<0.001 vs. BEAS-2B. A549 cells were transfected with oe-NC or oe-MNX1 and the (D) mRNA level and (E) protein expression of MNX1 using reverse RT-qPCR and western blotting, respectively. A549 cells were transfected with oe-MNX1 or sh-MNX1 or their negative controls and the (F) mRNA level and (G) protein expression of CCDC34 using RT-qPCR and western blotting, respectively. ***P<0.001 vs. oe-NC; ##P<0.01, ###P<0.001 vs. sh-NC. (H) As predicted from JASPAR website, MNX1 might bind to CCDC34 promoter. (I) Luciferase reporter assay was performed to verify the interaction between MNX1 and CCDC34 promoter and the luciferase activity was examined using dual-luciferase reporter system. ***P<0.001 vs. oe-NC. (J) ChIP assay was used to verify the interaction between MNX1 and CCDC34 promoter. ***P<0.001 vs. anti-IgG. MNX1, Motor neuron and pancreas homeobox 1; CCDC34, coiled-coil domain-containing 34; LUAD, Lung adenocarcinoma; GEPIA, Gene Expression Profiling Interactive Analysis; sh, short hairpin RNA; oe, overexpression; NC, negative control (empty vector); RT-qPCR, Reverse transcription-quantitative PCR; ChIP, chromatin immunoprecipitation.