MiR-1224-5p reverses gefitinib resistance in non-small-cell lung cancer cells by modulating RFX5/YAP1/HIF1α axis

Purpose: To investigate the molecular pathways by which miR-1224-5p modulate RFX5/YAP1/HIF1α pathway, thereby promoting gefitinib tolerance within non-small-cell lung cancer (NSCLC). Methods: To screen differentially expressed miR-1224-5p in NSCLC samples and predict its downstream target gene – RFX5 – by bioinformatics analysis, 60 NSCLC tissues and their corresponding paraneoplastic tissues were collected. Dual-luciferase assays were performed to verify the targeting relationship between miR-1224-5p and RFX5. Four NSCLC cell lines (A549, H1299, H2170, and H1975) and BEAS-2B normal lung epithelial cell lines were used for in vitro experiments. Co-immunoprecipitation (Co-IP) and Western blotting after cycloheximide (CHX) treatment were used to determine the regulatory interaction between YAP1 and HIF1α, with YAP1 modulating HIF1α protein stability. Results: In NSCLC, downregulation of miR-1224-5p was observed, which resulted in the decrease of RFX5 levels. This reduction in miR-1224-5p levels leads to a decrease in RFX5 levels. Furthermore, restoring miR-1224-5p expression in NSCLC cells made them more sensitive to gefitinib. In vitro, RFX5 elevates YAP1, which in turn boosts the stability of HIF1α. However, miR-1224-5p disrupts this mechanism by influencing the RFX5/YAP1/HIF1α pathway, thus mitigating resistance to gefitinib. Conclusion: The findings show that miR-1224-5p targets RFX5 to suppress YAP1 transcription, thereby diminishing HIF1α stability and overcoming gefitinib tolerance in NSCLC. These findings identify miR-1224-5p as a promising approach to address gefitinib tolerance in NSCLC.