ABSTRACT: Colorectal cancer (CRC) ranks among the most aggressive human malignancies and remains a leading cause of cancer mortality globally (Matsuda T. et al., Digestion, 2025). Despite advances in treatment, there is a critical need for novel therapeutic agents that combine high selectivity and minimal toxicity while effectively targeting the molecular mechanisms driving CRC progression. A growing body of evidence highlights the central oncogenic roles of microRNAs miR-21, miR-17, and miR-155 in CRC progression and malignancy (Fu F. et al., Transl Oncol, 2018; Sabry D. et al, Mol Cell Biochem, 2019; Yu W. et al., J Cancer, 2022; Wang D. et al., Cell Death Dis, 2022). Consequently, targeted suppression of these miRNAs through sequence-specific oligonucleotide-based therapeutics represents a promising strategy for simultaneously disrupting multiple carcinogenic pathways. Previous studies have demonstrated the potent anti-oncogenic efficacy of N-(methanesulfonyl)phosphoramidate (mesyl, µ-)-modified antisense oligonucleotides targeting miR-21, miR-155, and miR-17 across various tumor models (Miroshnichenko S. et al, PNAS, 2019; Patutina O. et al., PNAS, 2020, Gaponova (Miroshnichenko) S. et al., Cancers, 2022). In the present investigation, we aimed to elucidate the molecular mechanisms underlying the effects of these µ-ASOs in colorectal carcinoma cells. The study was conducted using human colorectal adenocarcinoma Caco-2 cells transfected with fully modified µ-ASOs targeting miR-21, miR-17, and miR-155 (oligonucleotides µ-21, µ-17 and µ-155, respectively). The major objectives of the investigation were: 1) to characterize the proteomic signatures induced by each individual µ-ASO, and (2) to assess the integrated proteomic response elicited by combined treatment with all three µ-ASOs. High-resolution proteomic profiling of µ-ASO-treated and control Caco-2 cells, following stringent contaminant filtration, identified 3.957 high-confidence proteins (≥2 unique peptides per protein). Pairwise comparisons between cells treated with control µ-ASO (µ-Scr); single miRNA-targeted µ-ASOs (µ-21, µ-17 and µ-155) or their triple combination (Combi) and control intact Caco-2 cells revealed a list of differentially expressed proteins (DEPs) showing at least a twofold change (log2FC ≥1, p < 0.05), including µ-Scr – 324 DEPs; µ-21 – 663 DEPs; µ-17 – 642 DEPs; µ-155 – 735 DEPs; Combi – 844 DEPs. Functional analysis of the identified DEPs demonstrated that all miRNA-targeted µ-ASOs consistently modulated core metabolic and protein synthesis pathways. In addition, each oligonucleotide also exhibited distinct effects on specific cancer-related processes: µ-21 predominantly influenced apoptosis, cell cycle regulation, proliferation, and DNA repair; µ-17 mainly modulated proliferation and chaperone-related stress responses; and µ-155 primarily affected intracellular transport, apoptotic signaling, and immune response regulation. Notably, the combined µ-ASO treatment produced a distinct, integrative effect—interconnecting these individual pathways into complex regulatory networks that collectively affected a wider spectrum of cellular functions. This work was supported by Russian Scientific Foundation (RSF), grant #19-74-30011
Svetlana Miroshnichenko 