The authors used bioinformatics analyses of downloaded breast cancer datasets and transcriptome data to screen for differentially expressed genes and candidate microRNA targets. RT-qPCR experiments were performed to compare miR-183-5p levels between normal breast epithelial cells and multiple breast cancer cell lines. The source reports that miR-183-5p expression was significantly elevated in breast cancer tissues relative to normal breast tissue, and was higher across the examined breast cancer cell lines than in normal epithelial cells. Among the cell lines tested, MDA-MB-231 cells showed the highest miR-183-5p expression (reported significance P < 0.0001 in the source).
Multiple online databases were combined with differential expression screening to predict potential miR-183-5p targets. The authors focused on Kelch-like protein 24 (KLHL24) as a candidate target gene. A dual‑luciferase reporter assay was used to test direct interaction between miR-183-5p and the 3′‑untranslated region (3′‑UTR) of KLHL24. The assay confirmed direct binding of miR-183-5p to the KLHL24 3′-UTR, supporting a post-transcriptional regulatory relationship (reported significance P < 0.001 in the source).
To evaluate the functional consequence of the miRNA–target interaction, TNBC cells were transfected with miR-183-5p–overexpressing plasmids. Western blot analysis demonstrated that overexpression of miR-183-5p significantly reduced KLHL24 protein levels (reported P < 0.0001). These data indicate that miR-183-5p negatively regulates KLHL24 expression at the protein level in this cellular context.
The authors conducted gain‑ and rescue‑of‑function experiments in MDA-MB-231 cells using four transfection conditions: NC-miR + NC-KLHL24 (controls), oe‑miR-183-5p + NC-KLHL24 (miR overexpression), NC-miR + oe‑KLHL24 (KLHL24 overexpression), and oe‑miR-183-5p + oe‑KLHL24 (combined overexpression). Cell proliferation was assessed by EdU incorporation, invasion by Transwell assay, and migration by wound healing assay. Overexpression of miR-183-5p markedly promoted TNBC cell proliferation, invasion, and migration (reported significance P < 0.001). Co‑overexpression of KLHL24 attenuated or reversed the pro‑proliferative and pro‑invasive/migratory effects of miR-183-5p, indicating that KLHL24 mediates, at least in part, the phenotypic consequences of miR-183-5p modulation.
The study examined core regulators and biochemical readouts related to ferroptosis. Western blotting was used to measure protein expression of GPX4, SLC7A11, and Nrf2—proteins implicated in ferroptosis resistance. Biochemical assays quantified reactive oxygen species (ROS), lipid peroxidation products (LPO), malondialdehyde (MDA), and reduced glutathione (GSH) levels.
According to the reported results, miR-183-5p overexpression led to upregulation of GPX4, SLC7A11, and Nrf2 protein expression (reported P < 0.05). Concurrently, miR-183-5p overexpression decreased intracellular ROS, LPO, and MDA levels (reported P < 0.0001) and increased GSH content (reported P < 0.0001). These changes are consistent with suppression of ferroptotic processes and enhancement of cellular antioxidant capacity. Overexpression of KLHL24 reversed the miR-183-5p–induced increases in GPX4, SLC7A11, and Nrf2 and normalized ROS, LPO, MDA, and GSH levels, indicating that KLHL24 counteracts the ferroptosis‑inhibitory effects of miR-183-5p in TNBC cells.
The article concludes that miR-183-5p is upregulated in breast cancer and TNBC cell lines and that its overexpression promotes malignant behaviors—proliferation, invasion, and migration—while inhibiting ferroptosis in TNBC cells. Mechanistically, these effects are mediated through direct negative regulation of KLHL24 by miR-183-5p; restoring KLHL24 expression attenuates both the pro‑tumorigenic phenotype and the ferroptosis suppression induced by miR-183-5p.
These findings identify a miR-183-5p/KLHL24 axis that influences TNBC cell behavior and ferroptosis‑related pathways, suggesting a potential molecular target for further preclinical investigation. The source reports statistical significance for expression and functional assays as noted above. Details on in vivo validation, patient cohort characteristics, or therapeutic testing were not reported in the source abstract and would require consultation of the full article for additional data.