Fig 2: Survival analysis. Kaplan-Meier survival curves for lung adenocarcinoma cases classified in terms of hub-gene expression. (A) ANGPTL7, (B) SLC6A4, (C) PTPRQ, (D) KCNA4 and (E) TEDC2. HR, hazard ratio.

Fig 3: Immunohistochemistry, CCK-8, colony formation, scratch assay, cell cycle analysis, Transwell invasion assay and cell adhesion assay. (A) Differential expression of SLC6A4 in tumor tissue (Ca) along with paraneoplastic healthy tissue (Para-Ca); scale bar, 20 µm. (B) Protein levels were assessed using western blot analysis. A549 cells were transfected with an empty vector as the negative control. GADPH was used as the internal control. ‘Flag_SLC6A4’ represents the exogenous expression of SLC6A4 and ‘Flag’ is a fusion tag. (C-H) SLC6A4 overexpression inhibits A549 cell growth, invasion, and migration. A549 cells which were transfected with an empty vector were used as the control. ‘Flag_SLC6A4’ represents the exogenous expression of SLC6A4. Cell proliferation was analyzed using (C) CCK-8 and (D) colony formation. (E) The effect of SLC6A4 overexpression on cell migration was assessed using scratch assays; scale bar, 100 µm. (F) Cell cycle distribution was determined using flow cytometry. (G) Cell invasion in the two groups was compared using Transwell assays. (H) Cell adhesion in both cell groups (magnification, ×40). Statistical significance was analyzed through unpaired t-tests (n=3). *P<0.05 and **P<0.01. OD, optical density; ns, no significance.

Fig 4: Bioinformatics validation. (A-E) Differential expression of the five hub genes between healthy and tumor tissue using data from The Cancer Genome Atlas and Genotype-Tissue Expression. (A) ANGPTL7, (B) SLC6A4, (C) PTPRQ, (D) KCNA4 and (E) TEDC2. (F) Differential expression of the five hub genes between healthy and tumor tissue in the GEO116959 dataset. LUAD, lung adenocarcinoma.