Dynamic Regulatory Mechanisms of the DHX9/ILF3 Complex During Lung Adenocarcinoma Cell Differentiation: A Single-Cell Transcriptomic Analysis

Background: The DExH-box helicase 9 (DHX9)/interleukin enhancer-binding factor 3 (ILF3) complex participates in RNA processing and post-transcriptional regulation, but its behavior during lung adenocarcinoma cell differentiation remains poorly defined, particularly at single-cell resolution. This study aimed to characterize the dynamic expression patterns and potential biological associations of the DHX9/ILF3 complex during epithelial-state transitions in lung adenocarcinoma using single-cell transcriptomic analysis. Methods: We analyzed publicly available single-cell RNA sequencing (scRNA-seq) datasets from the Gene Expression Omnibus (GEO), including the discovery dataset GSE131907 and the independent validation dataset GSE123902, to profile lung adenocarcinoma epithelial cells at single-cell resolution. The sequencing data were processed using a standard single-cell analytical workflow, including quality control, dimensionality reduction, clustering, pseudotime trajectory inference, co-expression analysis, and functional program scoring. To verify key transcriptional patterns identified in the single-cell dataset, we measured the mRNA levels of selected genes in A549 and H1975 cells using quantitative real-time PCR. Results: Single-cell analysis revealed multiple epithelial states, including normal-like epithelial cells, transitional tumor-associated states, and malignant cells. Across these states, core members of the DHX9/ILF3 complex, including DHX9, ILF3, ILF2, and HNRNPU, displayed coordinated yet dynamic expression patterns along pseudotime trajectories. DHX9 expression rose during the early stages of the trajectory and then declined, whereas ILF3 increased more gradually and remained relatively elevated in intermediate states. Correlation analysis indicated positive associations among key complex members at the single-cell level. Cells with higher DHX9/ILF3 complex activity exhibited stronger malignant transcriptional programs, including proliferation, stemness, and MYC-related signaling. External validation using the GSE123902 dataset reproduced the state-dependent expression patterns and DHX9–ILF3 correlation identified in the discovery dataset. qRT-PCR confirmed coordinated upregulation of DHX9, ILF3, ILF2, and HNRNPU in lung adenocarcinoma cell lines compared with normal epithelial cells, with similar patterns observed in A549 and H1975 cells. Conclusions: The DHX9/ILF3 complex exhibits coordinated yet dynamic activity during epithelial state transitions in lung adenocarcinoma. Its expression is associated with malignant transcriptional programs and is reproducible across independent single-cell datasets. These findings refine current understanding of RNA-binding protein–mediated regulation of tumor cell states and suggest the DHX9/ILF3 complex as a potential regulatory axis in lung adenocarcinoma.