Research on the mechanism of compound mylabris capsules regulating ferroptosis in lung adenocarcinoma based on bioinformatics and experimental validation

Background: Lung adenocarcinoma (LUAD) is characterized by insidious onset and early metastatic propensity. It is urgent to identify novel therapeutic agents with potent antitumor efficacy and low toxic side effects. This study aims to investigate the mechanism by which Compound Mylabris Capsules (CMC) regulates ferroptosis in LUAD and identify its active ingredients through integrated bioinformatics analysis and experimental validation. Materials and methods: The TCGA and GEO datasets related to LUAD were downloaded, and LUAD target genes were identified based on differentially expressed genes (DEGs) and weighted gene co-expression network analysis. Ferroptosis-related genes (FRGs) were obtained from the Genecards and FerrDb V2 databases, and the intersection with LUAD target genes yielded LUAD-FRGs intersection genes. A drug-active ingredient-target network was constructed to screen the active ingredients and targets of CMC. Common targets were obtained by taking the intersection of CMC targets and LUAD-FRGs intersection genes, and enrichment analysis was further performed. In vitro experiments were conducted to verify the effects of main active ingredients on A549 and H1650 cells. Single-cell RNA sequencing data were used to analyze the expression and distribution of common targets. Key genes were screened using Lasso regression, Boruta and RFE algorithms, and the expression levels, diagnostic and prognostic values of key genes were further analyzed. Molecular docking and molecular dynamics simulation were performed to validate the interactions between main active ingredients and key genes. Results: 2508 LUAD target genes were obtained after intersecting 6699 DEGs with 3829 key module genes. Further intersection of 1599 FRGs with LUAD target genes, 247 LUAD-FRGs intersection genes were identified. A total of 1074 CMC targets and the top three active ingredients (quercetin, kaempferol, β-sitosterol) were screened out. The intersection of CMC targets and LUAD-FRGs intersection genes yielded 31 common targets. Enrichment analysis demonstrated their significant involvement in biological processes such as Cell cycle, HIF-1 signaling pathway, VEGF signaling pathway, Cellular senescence and p53 signaling pathway. In vitro experiments showed that quercetin, kaempferol and β-sitosterol exerted inhibitory effects on A549 and H1650 cells. Single-cell RNA sequencing data analysis indicated that common targets were predominantly expressed in tumor cell subset. Machine learning identified CA9, EZH2, NQO1 and TERT as key genes, with their expression levels significantly elevated in the Tumor group. Additionally, ROC and univariate COX regression analyses suggested that these key genes possessed good diagnostic and prognostic values. Molecular docking and molecular dynamics simulation confirmed strong binding ability and stability between main active ingredients and key genes. Conclusion: CMC exerts antitumor effects in LUAD by regulating ferroptosis-related targets such as CA9, EZH2, NQO1 and TERT. This study reported for the first time that CMC alleviated LUAD by regulating ferroptosis.