Simple Summary: The role of miR-148a-3p in colorectal cancer (CRC) is still debated. In this study, the in vitro antineoplastic effect of miR-148a-3p overexpression in the CRC model is reported. The antitumor activity of miR-148a-3p occurs through apoptosis, mitochondrial impairment, lipid peroxidation, and ferroptosis sustained by the ACSL4/TFRC/Ferritin axis. Bioinformatic analysis and transfection experiments with miR-148a-3p mimics and inhibitors revealed that the cytotoxicity might be related to the downregulation of SLC7A11. These findings, extending knowledge on functional and molecular mechanisms, unveil the oncosuppressor role of miR-148a-3p, pointing out its potential as a diagnostic and therapeutic biomarker in CRC. Ferroptosis, an iron-dependent form of cell death, and dysregulated microRNA (miRNA) expression correlate with colorectal cancer (CRC) development and progression. The tumor suppressor ability of miR-148a-3p has been reported for several cancers. Nevertheless, the role of miR-148a-3p in CRC remains largely undetermined. Here, we aim at investigating the molecular mechanisms and regulatory targets of miR-148a-3p in the CRC cell death mechanism(s). To this end, miR-148a-3p expression was evaluated in SW480 and SW620 cells and normal colon epithelial CCD 841 CoN cells with quantitative real-time polymerase chain reaction (qRT-PCR). Data reported a reduction of miR-148a-3p expression in SW480 and SW620 cells compared to non-tumor cells (p < 0.05). Overexpression of miR-148a selectively inhibited CRC cell viability (p < 0.001), while weakly affecting normal CCD 841 CoN cell survival (p < 0.05). At the cellular level, miR-148a-3p mimics promoted apoptotic cell death via caspase-3 activation (p < 0.001), accumulation of mitochondrial reactive oxygen species (ROS) (p < 0.001), and membrane depolarization (p < 0.001). Moreover, miR-148a-3p overexpression induced lipid peroxidation (p < 0.01), GPX4 downregulation (p < 0.01), and ferroptosis (p < 0.01), as revealed by intracellular and mitochondrial iron accumulation and ACSL4/TFRC/Ferritin modulation. In addition, levels of SLC7A11 mRNA and protein, the cellular targets of miR-148a-3p predicted by bioinformatic tools, were suppressed by miR-148a-3p's overexpression. On the contrary, the downregulation of miR-148a-3p boosted SLC7A11 gene expression and suppressed ferroptosis. Together, these in vitro findings reveal that miR-148a-3p can function as a tumor suppressor in CRC by targeting SLC7A11 and activating ferroptosis, opening new perspectives for the rationale of therapeutic strategies through targeting the miR-148a-3p/SLC7A11 pathway.
