Article Figures & Data
Figures
- Figure 1.
Identification and characterization of differentially expressed long non-coding RNAs (lncRNAs) across lung adenocarcinoma (LUAD) stages. (A) Schematic overview of the analytical pipeline used for the objective identification and prioritization of prognostic lncRNAs in LUAD. lncRNA expression profiles from TCGA-LUAD (58 normal lung samples and 488 tumor samples) were analyzed in a stepwise manner, beginning with expression filtering, followed by stage-wise differential expression analysis, intersection-based candidate selection, and survival-based prioritization. (B) Flowchart depicting the stepwise filtering and selection of differentially expressed lncRNAs. From ~12,000 annotated lncRNAs, transcripts with low expression were removed by retaining only those with RPKM >1 in at least 20% of samples, yielding 959 ENSGs. These were then compared independently between normal samples and each tumor stage (Stage I-IV) using log2 (RPKM + 1) transformed values, Welch’s t-test, and Benjamini–Hochberg false discovery rate (FDR) correction (FDR <0.05, ≥2-fold change). This analysis identified 168 upregulated lncRNAs in Stage I, 155 in Stage II, 143 in Stage III, and 96 in Stage IV. Intersection analysis across all four stages yielded a core set of 68 lncRNAs consistently upregulated in all tumor stages relative to normal lung tissue, which were carried forward for subsequent analyses. (C) Selection of poor-prognosis lncRNAs from the 68 consistently upregulated candidates. Kaplan–Meier overall survival analysis was systematically performed for all 68 lncRNAs by stratifying patients into high and low-expression groups based on median expression. Five lncRNAs were selected for display based solely on predefined survival criteria: hazard ratio (HR) ≥1.5, log-rank p<0.05, and prior inclusion in the 68-lncRNA core set. These lncRNAs represent tumor-upregulated candidates whose higher expression is associated with poorer overall survival. (D) Identification of prognostic lncRNAs that are downregulated in LUAD tumors relative to normal tissue. Using the same statistical framework, stage-wise comparisons identified 92 downregulated lncRNAs in Stage I, 101 in Stage II, 109 in Stage III, and 78 in Stage IV. Intersection analysis across all four stages yielded 67 lncRNAs consistently downregulated in tumors. Kaplan–Meier survival analysis of these 67 lncRNAs identified four candidates whose higher expression was associated with significantly improved overall survival (HR ≤0.65, log-rank p<0.05), indicating potential tumor-suppressive or protective roles. These four lncRNAs’ survival plots are shown in Supplementary Figure 1. (E) Stage-wise expression of representative upregulated lncRNAs across LUAD progression. Samples are color-coded by stage: normal (green), stage I (yellow), stage II (blue), stage III (purple), and stage IV (red).
- Figure 2.
High expression of selected long non-coding RNAs (lncRNAs) correlates with poor overall survival in lung adenocarcinoma (LUAD) patients. (A-E) Kaplan–Meier overall survival plots for representative lncRNAs selected from the objectively defined candidate set described in Figure 1. Survival analysis was performed for all 68 lncRNAs consistently upregulated across all four LUAD stages relative to normal lung tissue. Patients were stratified into high- and low-expression groups based on median expression, and survival differences were assessed using the log-rank test, with corresponding hazard ratios (HRs) calculated. Panels (A-E) show lncRNAs that met predefined survival-based selection criteria, including significant association with overall survival (log-rank p<0.05) and elevated hazard ratios, indicating poorer prognosis in patients with higher expression. These lncRNAs were subsequently highlighted in Figure 1C as poor-survival–associated candidates. (F) Cytoplasmic–Nuclear Relative Concentration Index (CN-RCI) values for FAM83A-AS1, CYTOR, and MIR4435-2HG were obtained from the lncATLAS database (V24), which compiles RNA-seq-based nuclear and cytoplasmic fractionation data across multiple human cell lines. The dataset includes both lung-derived and non-lung cell lines representing diverse tissue origins, including A549 and NCI-H460 (lung cancer), GM12878 and K562 (hematopoietic/lymphoblastoid), H1-hESC (human embryonic stem cells), HeLa (cervical adenocarcinoma), HepG2 (hepatocellular carcinoma), HT1080 (fibrosarcoma), HUVEC (endothelial cells), MCF-7 (breast cancer), NHEK (normal human epidermal keratinocytes), SK-MEL-5 (melanoma), and SK-NSH (neuroblastoma). Violin plots depict the distribution of CN-RCI values for lncRNAs (blue) alongside protein-coding RNAs (orange) within each cell type. Positive CN-RCI values indicate cytoplasmic enrichment, whereas negative values indicate nuclear enrichment. The distributions shown reflect global localization tendencies across cell types rather than lung-specific localization. CN-RCI data were not available for AC245595.1 and AP001453.2, and these lncRNAs were therefore not included in the analysis.
- Figure 3.
Trend-based identification of long non-coding RNAs (lncRNAs) showing progressive expression changes across lung adenocarcinoma (LUAD) stages. (A) Rank-based trend analysis of lncRNA expression across ordered LUAD stages. Following the objective selection of 68 lncRNAs that were consistently upregulated across all four tumor stages relative to normal lung tissue (as described in Figure 1), a formal trend test was performed by treating tumor stage as an ordinal variable (Stage I < Stage II < Stage III < Stage IV). For each lncRNA, expression values from all LUAD tumor samples were ranked, and stage-wise average expression ranks were calculated. The strength and direction of monotonic expression change across stages were quantified using a rank-correlation–based trend coefficient, and statistical significance was assessed with FDR correction. Panel A displays lncRNAs that showed statistically significant monotonic trends (FDR-adjusted q<0.05), ordered according to the strength of the trend. Positive values indicate increasing expression with advancing tumor stage. (B) Stage-wise expression patterns of representative lncRNAs showing strong monotonic trends across LUAD progression. From the set of trend-positive lncRNAs identified in panel A, a subset of 12 lncRNAs with the strongest positive trends (≥0.8) was selected for visualization. Expression levels are shown across normal lung tissue and LUAD Stages I-IV, illustrating a progressive increase in expression with tumor advancement. Each data point represents an individual sample, and samples are grouped by clinical stage. This subset was chosen solely to facilitate visualization of the trend-based behavior and does not alter the conclusions derived from the full trend analysis. Sample sizes for each stage were as follows: Stage I (n=170), Stage II (n=70), Stage III (n=62), and Stage IV (n=17).
- Figure 4.
Expression and monotonic trend analysis of prognostic long non-coding RNAs (lncRNAs) across lymph-node status and tumor size in lung adenocarcinoma (LUAD). (A) Relative expression of selected lncRNAs across lymph-node status in LUAD patients (Normal, N0-N3). Sample sizes were as follows: Normal (n=58), N0 (n=316), N1 (n=180), N2 (n=72), and N3 (n=2). Statistical significance was assessed using a rank-based trend test with FDR correction, with significance levels indicated (*p<0.05; **p<0.01; ***p<0.001; ns, not significant) (see Supplementary Figure 3A), data are shown as mean±SD. (B) Relative expression of selected lncRNAs across primary tumor size categories (Normal, T1-T4). Sample sizes were: Normal (n=58), T1 (n=154), T2 (n=261), T3 (n=42), and T4 (n=18). Statistical significance is annotated as in panel A (see Supplementary Figure 3B). (C) Comparison of lncRNA expression between male and female LUAD patients. Sample sizes were: Male (n=223) and Female (n=265). Statistical comparisons were performed using a two-sided Welch’s t-test. No statistically significant sex-specific differences were observed for any of the analyzed lncRNAs; ns: not significant.
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