MiR-494-5p suppresses colorectal cancer metastasis by multi-level targeting of KAT2B, a novel mechanism disrupting KAT2B enhancer-promoter looping, and inhibiting c-JUN-driven pro-metastatic properties.

backgroundMetastatic progression is the primary cause of death in colorectal cancer (CRC). However, the molecular networks governing CRC metastasis remain incompletely understood, representing a critical knowledge gap. MicroRNAs (miRNAs) are key post-transcriptional regulators frequently dysregulated in CRC, yet their role in metastasis, particularly via non-canonical mechanisms, remains largely underexplored.

methodsIn this study, we identify miR-494-5p as a multi-layer master-regulator acting via several, in part previously undescribed, targeting mechanisms towards KAT2B and key molecular processes in CRC metastasis, using different in vitro and in vivo models, chromosome conformation capture, chromatin and RNA immunoprecipitation, GST- and RNA pull-down, gene editing, and further methods to delineate epigenetic miR-action on promoter enhancer looping. Besides, classical methods delineating translational and transcriptional miR-target interactions in cultured CRC and validations in CRC tissue datasets were applied.

resultsLow expression of miR-494-5p correlates with poor patient survival, and miR-overexpression inhibits cell migration, EMT, invasion, and metastasis in vivo. Mechanistically, we identify the histone acetyltransferase KAT2B as a multi-level target of miR-494-5p. Importantly, besides direct classical miR-targeting at the mRNA and gene promoter, we unveil a novel epigenetic mechanism whereby nuclear miR-494-5p silences KAT2B transcription by recruiting Argonaute proteins and altering histone modifications (increasing H3K27me3, decreasing H3K27ac/H3K4me1) at a specific KAT2B enhancer, to which miR-494-5p binds. This leads to the disruption of the mediator complex subunit 1 (MED1)-p300-RNA polymerase II (RNAP II) coactivator complex and dissolution of enhancer-promoter looping, as confirmed by Chromosome Conformation Capture (3C). Downstream, KAT2B epigenetically activates the transcription of the AP-1 component c-JUN by maintaining H3K27 acetylation at its locus. Crucially, KAT2B-induced c-JUN transcriptionally upregulates u-PAR/PLAUR, a key effector promoting several aspects of metastasis. c-JUN knockdown phenocopies the anti-EMT action of KAT2B loss or miR-494-5p. Importantly, c-JUN overexpression rescues the metastatic phenotype suppressed by miR-494-5p or KAT2B knockdown in vitro and in vivo, and this is dependent on u-PAR/PLAUR upregulation.

conclusionsWe delineate a novel miR-494-5p/KAT2B/c-JUN/PLAUR regulatory axis, whereby miR-494-5p functions as a multi-level metastatic suppressor by orchestrating translational, transcriptional, and epigenetic silencing of oncogenic KAT2B. This suppresses c-JUN-dependent cascades including u-PAR/PLAUR, suggesting a novel, miR-orchestrated master-regulatory axis in CRC progression and metastasis.