Differential Transcriptome Analysis of Lung Squamous Cell Carcinoma Under Different RUNX3 Expression Patterns

Runt‐related transcription factor 3 (RUNX3) functions as a tumor suppressor gene in numerous cancer types, including non‐small cell lung cancer, with its hypermethylation status predicting worse patient prognosis. However, any discrepancy in gene expression profiles in lung squamous cell carcinoma (LUSC) patients with different RUNX3 expression patterns remains unclear. In this study, RNA sequencing analysis was used to examine the transcriptomic differences in LUSC samples depending on their RUNX3 expression status.

Immunohistochemistry staining was used to determine the RUNX3 protein expression patterns in tumor samples. Tumor RNA was extracted from 63 LUSC cases and underwent RNA sequencing. Read counts were analyzed using Spearman's correlation, limma differential expression with Benjamini‐Hochberg false discovery rate (BH‐FDR) control, and gene set variation analysis (GSVA).

In our LUSC cohort, 31 (49.21%) patients were classified as RUNX3_Positive (RUNX3_Pos) and 32 (50.79%) were classified as RUNX3_Negative. RUNX3 mRNA expression levels were significantly higher in the RUNX3_Pos group. Using limma with BH correction, no genes passed the BH‐FDR < 0.05 threshold between the groups, suggesting subtle gene‐level differences. Therefore, we reported an exploratory nominal gene list (p < 0.05 and |log₂ fold‐change| > 1; 131 upregulated and 57 downregulated) for hypothesis generation only. Spearman's correlation identified 5047 positively and 25 negatively correlated mRNAs with RUNX3. GSVA suggested exploratory pathway activity patterns, with 57 pathways showing nominally higher and nine pathways showing nominally lower GSVA scores in RUNX3_Pos's tumors.

The RUNX3‐defined groups showed no BH‐FDR‐significant differentially expressed genes, but exhibited exploratory, nominal pathway activity patterns. These results generate testable hypotheses on RUNX3‐related biological programs in LUSC for future validation.