Biological Performance and Molecular Mechanisms of Mesyl MicroRNA-Targeted Oligonucleotides in Colorectal Cancer Cells

Colorectal cancer (CRC) remains one of the most aggressive and therapeutically resistant malignancies worldwide. This study examined the molecular mechanisms underlying the anti-oncogenic activity of methanesulfonyl phosphoramidate-modified antisense oligonucleotides (µ-ASOs) targeting miR-21, miR-17, and miR-155, which represent critical oncogenic drivers in CRC. Using human colorectal adenocarcinoma Caco-2 cells transfected with either individual µ-ASOs or their triple combination, we assessed target miRNA downregulation, antiproliferative and anti-migratory activities, and performed extensive proteomic profiling. Protein–protein interaction network analysis of differentially expressed proteins (DEPs) revealed that, beyond modulation of core metabolic processes, each µ-ASO exhibited distinct effects: µ-21 predominantly affected apoptosis, cell cycle, and DNA repair; µ-17 influenced proliferation and chaperone responses; and µ-155 modulated intracellular transport and immune regulation. Combination treatment elicited a unique proteomic signature partially overlapping with monotherapies. The proteomic analysis revealed several validated and putative miRNA-targeted DEPs, including both established and novel candidates in the CRC context: RPL31, CCT2, and CDC37 (miR-21); DNM2, SNRPN, NUP98, and NUP85 (miR-17); as well as RPL17 (miR-155). Expression of these targets correlated with favorable clinical outcomes in CRC patients. This work provides the first comprehensive mechanistic insight into antisense oligonucleotide-mediated miRNA suppression in Caco-2 colorectal adenocarcinoma cells and expands the miRNA target landscape.