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Background Based on bioinformatics analysis and experimental validation, we investigated the expression, clinical significance, immune infiltration, and potential signaling pathways of miR-3940-5p in lung adenocarcinoma (LUAD). Methods 521 LUAD tissue samples and 46 normal lung tissue samples from The Cancer Genome Atlas (TCGA) database. We evaluated the relationship between clinical features and miR-3940-5p expression using Kruskal–Wallis, Wilcoxon sign-rank, and logistic regression, explored the relationship between miR-3940-5p expression and the prognosis of LUAD patients using Kaplan–Meier survival curve analysis. Several databases were used to identify miRNA targets. MiR-3940-5p target genes were analyzed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The significant role of miR-3940-5p in function was evaluated using immune infiltration analysis. LUAD cell lines were tested for miR-3940-5p expression using QRT-PCR. Results There was a significant association between high miR-3940-5p expression in LUAD and T stage (P=0.005), pathologic stage (P=0.047), race (White vs Asian & Black or African American) (P=0.041), residual tumor (P=0.043), and anatomic neoplasm subdivision2 (P=0.030). MiR-3940-5p expression predicted poor overall survival (HR: 1.35; 95% CI: 1.01–1.81; P=0.045), disease-specific survival (HR: 1.53; 95% CI: 1.05–2.23; P=0.026), and progression-free survival (HR: 1.35; 95% CI: 1.03–1.77; P=0.032). BAP1, BBS1, CCR2, KCNE3, PEBP1, and RABL2A were all associated with poor OS in LUAD patients with low miR-3940-5p expression levels. According to GO and KEGG analyses, miR-3940-5p may play a role in LUAD development by regulating pathways such as measles, PI3K-Akt signaling pathway, and p53 signaling pathway. There was a correlation between the expression level of miR-3940-5p and immune infiltration. LUAD cell lines showed significantly higher levels of miR-3940-5p than Beas-2B cells. Conclusion A high expression of miR-3940-5p is significantly associated with a poor prognosis in patients with LUAD, suggesting that it could be used as a prognostic biomarker.

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