Dysregulated expression patterns of hsa-miR-9-5p and hsa-let-7a-5p are associated with altered NFAT5, ARID1A, and ZFP36 expression in NSCLC cell lines

Abstract

Objectives: This study aimed to evaluate the expression patterns of hsa-miR-9-5p and hsa-let-7a-5p together with nuclear factor of activated T-cells 5 (NFAT5), AT-rich interacting domain-containing protein 1A (ARID1A), and zinc finger protein 36 (ZFP36) in non-small cell lung cancer (NSCLC) cell lines and to externally contextualize these findings using ENCORI/starBase transcriptomic data.

Materials and methods: The A549 and H1299 NSCLC cell lines and the non-tumorigenic bronchial epithelial BEAS-2B cell line were cultured under standard conditions. Total ribonucleic acid (RNA), including small RNA fractions, was isolated and converted into complementary deoxyribonucleic acid for microRNA (miRNA) and messenger RNA (mRNA) analyses. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses were performed using SYBR Green chemistry. qRT-PCR was performed to determine the expression levels of hsa-miR-9-5p, hsa-let-7a-5p, NFAT5, ARID1A, and ZFP36. U6 and glyceraldehyde-3-phosphate dehydrogenase were used as endogenous controls for miRNA and mRNA analyses, respectively. Relative expression was calculated using the 2-ΔΔCt method, and statistical analyses were performed using ΔCt values. ENCORI/starBase was used to examine tumor-normal expression patterns and miRNA-mRNA co-expression relationships in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) cohorts.

Results: The hsa-miR-9-5p expression was significantly increased in A549 and H1299 cells compared with BEAS-2B cells, whereas hsa-let-7a-5p expression was significantly reduced. NFAT5 expression was markedly upregulated in both NSCLC cell lines, while ARID1A and ZFP36 expression levels were decreased, particularly in A549 cells. ENCORI/starBase analyses partially supported the in vitro findings, showing higher hsa-miR-9-5p expression and lower hsa-let-7a-5p and ZFP36 expression in lung cancer samples. Co-expression analysis further showed a weak but significant inverse correlation between hsa-miR-9-5p and ZFP36 in both LUAD and LUSC.

Conclusion: The findings demonstrate a coordinated expression profile in NSCLC cell lines characterized by increased hsa-miR-9-5p and NFAT5 expression together with reduced hsa-let-7a-5p, ARID1A, and ZFP36 expression. ENCORI/starBase data provide external support, particularly for the hsa-miR-9-5p/ZFP36 relationship. However, these findings should be interpreted as association-level evidence, and further functional studies are required to validate direct miRNA-mRNA regulatory interactions.

Cite this article as: Kızılkaya YT, Yıldız Ünal P. Dysregulated expression patterns of hsa-miR9-5p and hsa-let-7a-5p are associated with altered NFAT5, ARID1A, and ZFP36 expression in NSCLC cell lines. D J Med Sci 2026;12(1):20-35. doi: 10.5606/fng.btd.2026.239.