Mechanistic study of miR-124-3p targeting ATPase copper-transporting alpha to regulate copper homeostasis and cuproptosis sensitivity in hepatocellular carcinoma

Objective This study aimed to investigate the role of microRNA-124-3p (miR-124-3p)-mediated regulation of ATPase copper-transporting alpha (ATP7A) in hepatocellular carcinoma (HCC) progression and cuproptosis sensitivity, providing new insights into copper metabolism-based therapeutic strategies. Methods and Results Bioinformatics analyses were performed to evaluate ATP7A expression, prognostic significance, and potential biological functions in HCC. ATP7A expression was significantly increased at both mRNA and protein levels in HCC tissues, and elevated ATP7A expression was associated with unfavorable overall survival. Further analysis identified miR-124-3p as a potential upstream regulator of ATP7A, which was confirmed by dual-luciferase reporter assays. qRT-PCR and Western blot analyses demonstrated reduced miR-124-3p expression and increased ATP7A expression in HCC cells. Functional assays revealed that miR-124-3p inhibition promoted HCC cell proliferation, migration, and invasion, whereas ATP7A silencing partially abolished these effects. Mechanistically, ATP7A knockdown increased intracellular copper accumulation, enhanced reactive oxygen species (ROS) production, promoted FDX1 and LIAS expression, and facilitated DLAT oligomerization, accompanied by G1-phase cell cycle arrest and apoptosis. Moreover, ATP7A depletion rescued the malignant phenotypes and cuproptosis-related alterations induced by miR-124-3p inhibition. Conclusion Our findings demonstrate that the miR-124-3p/ATP7A axis regulates copper homeostasis and cuproptosis sensitivity in HCC. Downregulation of miR-124-3p enhances ATP7A expression, promotes copper efflux, and reduces cuproptosis susceptibility, thereby facilitating HCC progression. Targeting this regulatory axis may represent a potential strategy for cuproptosis-based HCC therapy.