Computational Analysis Links BUB1 and Noncoding RNA Network to Lung Adenocarcinoma Outcomes

In an in silico multi-omics investigation of lung adenocarcinoma (LUAD), researchers identified an oncogenic regulatory axis centered on the mitotic checkpoint kinase BUB1, driven by transcription factor E2F1 and a competing endogenous RNA (ceRNA) network comprising long noncoding RNA TMPO-AS1 and microRNA hsa-let-7b-5p. Analysis across TCGA-based platforms demonstrated that elevated BUB1 transcript levels significantly correlated with reduced overall survival in LUAD patients (HR = 1.86, CI 1.56–2.22, P = 2.6e−12). This survival decrement was pronounced in smokers (HR = 1.76, CI 1.35–2.29, P = 2.5e−05), while showing no significant association in non-smokers (HR = 1.61, CI 0.88–2.92, P = 0.12). Gender-stratified analyses of smokers confirmed poorer outcomes in both males (HR = 1.6, P = 0.0081) and females (HR = 1.87, P = 0.0021). In contrast, survival analyses in lung squamous cell carcinoma revealed no significant prognostic link, supporting BUB1’s role as a disease-specific classifier.

Differential expression analyses across genomic repositories documented significant BUB1 upregulation in LUAD tumors compared to normal tissues. UALCAN and OncoDB revealed significant BUB1 overexpression with p-values below 1e−12 and 8.1e−74, respectively, corroborated by ENCORI, Lung Cancer Explorer, and GEPIA2. In normalized TCGA-LUAD samples (normal n = 59, tumor n = 59), Wilcoxon rank-sum testing confirmed consistent BUB1 upregulation in tumors (P = 2.63e−16), while TCGAnalyzeR single-cell transcriptomics showed a 2.46 log2-fold increase in tumor cells. Clinical analyses via UALCAN indicated that BUB1 expression increased across advanced stages, showed higher levels in males, and was markedly elevated in smokers and cases with advanced nodal metastasis. TNMplot data confirmed significant upregulation in tumor and metastatic tissues compared to normal tissues (P = 3.82e−106). Furthermore, BUB1 upregulation was significantly associated with TP53 mutations in UALCAN (P < 1e−12) and TIMER2.0 (P < 2.2e−16), while CancerProteome evaluations revealed increased BUB1 protein levels and a strong positive correlation between RNA and protein abundance (R = 0.713).

To identify upstream transcriptional regulators of BUB1, the researchers queried the Enrichr TRRUST database, revealing strong associations with several candidate transcription factors. Correlation analysis using ENCORI confirmed positive associations between BUB1 and multiple factors, including E2F4 (R = 0.219), MYC (R = 0.201), YBX1 (R = 0.413), TFDP1 (R = 0.442), E2F3 (R = 0.548), and E2F1 (R = 0.664), with E2F1 exhibiting the strongest correlation. Promoter sequence scanning from 2 kb upstream to 100 bp downstream of the BUB1 transcription start site using the JASPAR 2022 motif (MA0024.3) revealed multiple high-confidence E2F1 binding sites with relative scores between 0.80 and 0.92 clustered 0.6 to 1.9 kb upstream. Kaplan-Meier survival curves linked E2F1 upregulation to poor overall survival in LUAD (HR = 1.63, CI 1.37–1.94, P = 2.2e−08) and LUAD smokers (HR = 1.49, CI 1.14–1.93, P = 0.0028). Normalized sample comparisons (n = 59 each) confirmed E2F1 overexpression in tumors (P = 5.05e−08), while TNMplot showed its elevated expression in metastatic tissues (P = 1.47e−25).

Investigation of post-transcriptional regulation utilized the miRNet database, identifying 17 microRNAs associated with both BUB1 and E2F1. Differential expression screening through UALCAN established that five of these microRNAs—hsa-mir-126-3p, hsa-let-7g-5p, hsa-miR-98-5p, hsa-mir-29c-3p, and hsa-let-7b-5p—were significantly downregulated in LUAD tissues. Among these, hsa-let-7b-5p and hsa-miR-29c-3p showed the strongest negative correlations with BUB1 (R = −0.366 and R = −0.426) and E2F1 (R = −0.365 and R = −0.347) in ENCORI. Kaplan-Meier Plotter analysis linked lower expression of hsa-let-7b-5p (HR = 0.71, CI 0.53–0.95, P = 0.021) and hsa-miR-29c-3p (HR = 0.54, CI 0.37–0.79, P = 0.0012) to poor overall survival. Receiver operating characteristic analysis in CancerMIRNome demonstrated high prognostic accuracy for hsa-let-7b-5p (AUC = 0.89, CI 0.85–0.92) compared to moderate accuracy for hsa-miR-29c-3p (AUC = 0.67, CI 0.58–0.77). In normalized datasets (n = 46 each), hsa-let-7b-5p was significantly downregulated in tumor tissues (P = 0.025), showing consistent repression across smoking history, male sex, advanced stages, and nodal metastasis.

Computational hybridization assessments using miRWalk and RNA22v2 demonstrated strong binding affinities for hsa-let-7b-5p with BUB1 (−21.8 kcal/mol) and E2F1 (−22.4 kcal/mol), yielding folding energies of −13.89 kcal/mol and −13.90 kcal/mol, respectively. To characterize the long noncoding RNA components of the ceRNA network, the authors identified the top ten lncRNAs associated with BUB1 in Enrichr. Differential expression analysis in UALCAN revealed that five of these lncRNAs were significantly upregulated in LUAD, with TMPO-AS1 showing the strongest correlation with the network components. TMPO-AS1 was positively correlated with BUB1 (R = 0.632) and E2F1 (R = 0.581), while exhibiting an inverse correlation with hsa-let-7b-5p (R = −0.277). Thermodynamic folding analysis using RNA22v2 predicted a stable TMPO-AS1/hsa-let-7b-5p heteroduplex with a folding energy of −12.40 kcal/mol. These computational metrics suggest that TMPO-AS1 acts as a molecular sponge that sequesters hsa-let-7b-5p, diminishing microRNA-mediated suppression of BUB1. Concentric radar targetgrams from TNMplot corroborated this mechanism, showing marked upregulation of BUB1 and E2F1 accompanied by moderate TMPO-AS1 elevation in lung tumor samples.

Prognostic analysis of TMPO-AS1 in Kaplan-Meier Plotter revealed that elevated lncRNA expression significantly correlated with adverse patient outcomes. In univariate assessments, high TMPO-AS1 levels were associated with reduced overall survival (HR = 1.5, CI 1.29–1.74, P = 7.2e−08), first progression survival (HR = 1.9, CI 1.52–2.38, P = 8.1e−09), and post-progression survival (HR = 1.77, CI 1.32–2.39, P = 0.00013). In LUAD patients, TMPO-AS1 overexpression correlated with poor overall survival (HR = 2.16, CI 1.69–2.76, P = 4.1e−10), with significant hazard ratios in stage 1 (HR = 2.65, P = 4.4e−06), stage 2 (HR = 2.1, P = 0.0061), males (HR = 1.95, P = 9.1e−05), and females (HR = 2.83, P = 7.2e−07). Detrimental survival associations persisted among LUAD smokers (HR = 2.34, CI 1.41–3.88, P = 0.00066), male smokers (HR = 2.33, P = 0.0046), and female smokers (HR = 3.13, P = 0.016). Normalized sample comparisons (n = 59 each) confirmed TMPO-AS1 overexpression in tumors (P = 1.05e−13), with UALCAN indicating higher expression in males, smokers, and advanced cancer stages.

Functional enrichment and cell cycle profiling linked BUB1 expression to mitotic checkpoints and aggressive cellular behaviors. Querying Cyclebase 3.0 revealed elevated BUB1 expression specifically during the M phase of the cell cycle, ranking 15th among surveyed genes. Analysis of single-cell functional states via CancerSEA demonstrated significant positive correlations between BUB1 expression and DNA damage (R = 0.55), cell cycle regulation (R = 0.53), proliferation (R = 0.47), DNA repair (R = 0.42), invasion (R = 0.40), and epithelial-mesenchymal transition (R = 0.37). In ENCORI co-expression assessments, BUB1, E2F1, and TMPO-AS1 exhibited positive correlations with critical cell cycle regulators, whereas hsa-let-7b-5p displayed inverse correlations. Specifically, BUB1 correlated strongly with CDK1 (R = 0.88), CDK2 (R = 0.72), CCNE1 (R = 0.72), and CCNB1 (R = 0.84). E2F1 similarly correlated with CDK2 (R = 0.649), CDK1 (R = 0.631), CCNE1 (R = 0.681), and CCNB1 (R = 0.649), while TMPO-AS1 correlated with CDK2 (R = 0.646), CDK1 (R = 0.626), CCNE1 (R = 0.522), and CCNB1 (R = 0.646), underscoring coordinate involvement in S and M phase checkpoint transitions.

Evaluation of pathological stage expression trends in GSCA showed a steady increase in BUB1 expression across all LUAD stages without fluctuations, while TMPO-AS1 displayed progressive upregulation. In comparison, E2F1 rose from Stage I to Stage III, MKI67 increased through Stage II before dipping in Stage III, and TP53 peaked in Stage III before dropping in Stage IV. Composite Gene Set Variation Analysis scores were higher in tumor tissues than normal lung tissues, trending upward with advancing disease stages. To explore immunological associations, single-cell immune mapping in SPRING viewer revealed broad BUB1 distribution across non-small cell lung cancer immune compartments, alongside moderate E2F1 and TMPO-AS1 signals. In GSCA correlation analyses, CD4⁺ T cells demonstrated the strongest negative correlation with BUB1, E2F1, and TMPO-AS1 among all evaluated immune types. Furthermore, the combined BUB1–E2F1–TMPO-AS1 GSVA score showed a robust inverse correlation with CD4⁺ T cell infiltration (R = −0.56, FDR = 4.2e−42), validated in TIMER2.0 across multiple estimation algorithms.

Molecular docking was conducted using AutoDock Tools 1.5.7 against the BUB1 crystal structure (PDB ID: 6F7B) to investigate potential therapeutic interactions with selective inhibitors, chemotherapeutics, and natural compounds. Docking generated ten conformations per ligand, with top-ranked binding affinities evaluated for BAY-1816032 (−8.0 kcal/mol), paclitaxel (−8.2 kcal/mol), docetaxel (−7.6 kcal/mol), quercetin (−7.4 kcal/mol), and hesperidin (−9.4 kcal/mol). Hesperidin exhibited the highest binding affinity, surpassing both standard chemotherapeutic agents and the selective inhibitor. Visualization using Discovery Studio Visualizer showed that hesperidin established an extensive interaction network comprising multiple conventional hydrogen bonds and van der Waals contacts with crucial residues in the BUB1 active site, whereas BAY-1816032 primarily engaged through π-alkyl contacts and hydrogen bonds. In separate docking simulations assessing tobacco-derived carcinogens, nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) demonstrated binding affinities of −4.7 kcal/mol and −5.5 kcal/mol with BUB1, respectively, suggesting molecular interactions that might contribute to cell-cycle signaling dysregulation in smokers.

Extracellular liquid biopsy feasibility was evaluated using EVAtlas and EVmiRNA 2.0, which confirmed detectable levels of hsa-let-7b-5p across various human biofluids, including serum, plasma, breast milk, amniotic fluid, and cord blood. In discussing these findings, the authors emphasized key methodological limitations inherent to their study design. Specifically, the entire investigation relied on computational modeling, public omics databases, and bioinformatic predictions without prospective clinical validation or wet-lab experimental testing. The researchers noted that while public repositories provide validated datasets, future research must incorporate prospective clinical cohorts and rigorous experimental validation to confirm the biological reality of the proposed BUB1/E2F1/TMPO-AS1/hsa-let-7b-5p axis. They also highlighted that experimental assays are necessary to establish the therapeutic efficacy and safety profile of hesperidin as a potential low-toxicity BUB1 inhibitor in lung adenocarcinoma. Consequently, the identified ceRNA network serves as a computational framework for potential biomarker discovery, awaiting translational and laboratory verification before any clinical application.

Subject of Research: BUB1 expression and regulatory ceRNA network in lung adenocarcinoma

Article Title: hsa-let-7b-5p-associated BUB1/TMPO-AS1 ceRNA axis identified as a potential biomarker in lung adenocarcinoma

Article References: Baweja, B., Vats, P., Saini, C., Kumar, A., & Nema, R. (2025). hsa-let-7b-5p-associated BUB1/TMPO-AS1 ceRNA axis identified as a potential biomarker in lung adenocarcinoma. Cell Division, 21(1), Article 2. https://doi.org/10.1186/s13008-025-00173-x

Image Credits: AI Generated

DOI: 10.1186/s13008-025-00173-x

Keywords: BUB1, lung adenocarcinoma, ceRNA network, hsa-let-7b-5p, TMPO-AS1, E2F1, computational oncology, biomarkers, hsa-let-7b-5p-associated, ceRNA, axis, identified