Simple SummaryBreast cancer has a high prevalence in females worldwide, with persistent challenges in its treatment. Gold nanoparticles (AuNPs) have gained popularity for their anti-tumorigenic role, but their therapeutic function in microRNA (miRNA) regulation has never been explored. This study determines the potential of chemically engineered AuNPs against tumorigenic protein matrix metallopeptidase 9 (MMP-9). The data shows that in breast cancer cells, microRNA-204-5p is a direct regulator of MMP-9. AuNPs inhibit MMP-9 expression through the upregulation of microRNA-204-5p in a dose-dependent manner. Moreover, AuNPs also inhibit nuclear transcription factor, NF-κB activation in breast cancer cells transfected with anti-hsa-miR-204. In short, AuNPs are stable and non-toxic to breast cancer cells. AuNPs inhibit MMP-9 expression and NF-κBp65 induction and upregulate microRNA-204. These novel therapeutic potentials of AuNPs on breast cancer cells provide insight that AuNPs inhibit carcinogenic activity via microRNAs.AbstractObjective: Breast cancer (BC) is the most common malignancy in females globally. Matrix metalloproteinase-9 (MMP-9) is crucial to the invasion, progression and spread of BC. Gold nanoparticles (AuNPs) have an anti-tumorigenic role, but their therapeutic role in microRNAs (miRNAs) regulation has not been explored. This study determined the potential of AuNPs against MMP-9 overexpression/production and miRNA-204-5p regulation in BC cells. Methods: AuNPs were newly engineered, and their stability was analyzed using the zeta potential, polydispersity index, surface-plasmon-resonance peak and transmission electron microscopy. A bioinformatics algorithm was used to predict the pairing of miRNA in the 3′untranslated-region (3′UTR) of MMP-9 mRNA. TaqMan assays were carried out to quantify miRNA and mRNA, whereas MMP-9-specific immunoassays and gelatin zymography were used to determine protein secretion and activity. The binding of miRNA in MMP-9 mRNA 3′UTR was verified by luciferase reporter clone assays and transfection with anti-miRNAs. In addition, NF-κBp65 activity was determined and confirmed with parthenolide treatment. Results: Engineered AuNPs were highly stable and spherical in shape, with a mean size of 28.3 nm. Tested in MCF-7 BC cells, microRNA-204-5p directly regulates MMP-9. AuNPs inhibit PMA-induced MMP-9 mRNA and protein via hsa-miR-204-5p upregulation. Anti-miR-204 transfected MCF-7 cells demonstrated enhanced MMP-9 expression (p< 0.001), while AuNPs treatment attenuated MMP-9 expression in a dose-dependent manner (p< 0.05). Moreover, AuNPs also inhibit PMA-induced NF-κBp65 activation in anti-hsa-miR-204 transfected MCF-7 cells. Conclusion: Engineered AuNPs were stable and non-toxic to BC cells. AuNPs inhibit PMA-induced MMP-9 expression, production and activation via NF-κBp65 deactivation and hsa-miR-204-5p upregulation. These novel therapeutic potentials of AuNPs on stimulated BC cells provide novel suggestions that AuNPs inhibit carcinogenic activity via inverse regulation of microRNAs.