Unraveling the miR-144-3p/PUMA pathway: a novel regulator of FDX1-mediated cuproptosis in colorectal cancer.

[PURPOSE] Cuproptosis represents a novel form of cell death, predominantly driven by excessive intracellular accumulation of Cu. The mechanisms underlying cuproptosis in colorectal cancer (CRC) remain unknown. This study aims to investigate the underlying mechanisms in CRC, offering a novel therapeutic strategy for its treatment.

[METHODS] Transcriptomic datasets from The Cancer Genome Atlas (TCGA) databases were analyzed using R-software (v4.3.1) to quantify miR-144-3p expression in CRC. In vitro, CCK-8, EdU, colony formation assays, and Transwell assays were used to assess the proliferative capacity, migratory potential, and invasive properties of CRC under miR-144-3p modulation. Western blotting (WB) further linked miR-144-3p expression to modulation of cuproptosis-related pathways. RNA immunoprecipitation (RIP) and dual-luciferase reporter assays were performed to validate miR-144-3p binding to the 3′-UTR of mRNA. Co-IP and Immunofluorescence (IF) assays confirmed PUMA-FDX1 binding. DLAT oligomerization was analyzed by WB and IF experiments. The ubiquitination level of FDX1 was detected via Co-IP. In vivo, tumor xenograft experiments using HCT116 and HCT116 cell lines were performed in nude mice.

[RESULTS] In this study, miR-144-3p, a recently identified microRNA, was found to be aberrantly upregulated in CRC. It significantly promoted the proliferation, migration, and invasion of CRC both in vitro and in vivo. Mechanistically, miR-144-3p directly bound to the 3′-UTR of mRNA, thereby inhibiting PUMA expression and function, which restrained cell death. Furthermore, PUMA induces cuproptosis by enhancing the function of FDX1 through the directly binding with its R155 site, which promotes the oligomerization of lipoylated DLAT/DLST and reduces LIAS expression. In addition, we identified the way in which FDX1 is depleted in the process of cuproptosis: the binding between PUMA and FDX1 raised the ubiquitination level at the K182 site, leading to the further degradation of FDX1.

[CONCLUSIONS] Our findings delineate a miR-144-3p/PUMA regulatory axis controlling cuproptosis homeostasis in CRC. PUMA acts as a functional regulator of cuproptosis, activating copper-dependent cell death by binding FDX1, thereby enhancing DLAT/DLST oligomerization and suppressing LIAS expression. This axis may offer a promising new approach and target for the treatment of CRC.

[SUPPLEMENTARY INFORMATION] The online version contains supplementary material available at 10.1007/s13402-025-01093-2.