Development of gemcitabine-modified miR-15a as a novel, multimodal treatment strategy to overcome 5-FU and oxaliplatin resistance in colorectal cancer

Background Resistance to 5-fluorouracil (5-FU)-based chemotherapy is a major clinical obstacle in colorectal cancer (CRC), highlighting the urgent need to overcome established resistance mechanisms. MicroRNA-based therapeutics have emerged as compelling candidates in this context, given their inherently pleiotropic mode of action; however, their clinical translation remains hindered by poor stability and suboptimal delivery.

To address these limitations, Gem-miR-15a, a unique gemcitabine-modified tumor-suppressor microRNA-15a was designed to synergistically integrate the tumor-suppressive activity of miR-15a with the chemotherapeutic potency of gemcitabine into a single molecular entity. Therapeutic efficacy of Gem-miR-15a was evaluated across a spectrum of preclinical models, including parental and drug-resistant CRC cell lines, 3D tumor spheroids, patient-derived organoids and in vivo metastatic models. Cell viability, apoptosis and cell cycle analyses were performed, along with RNA sequencing and protein validation. Statistical analyses were conducted using Student’s t-test or two-way ANOVA with mixed effects, and data were presented as mean ± SD.

Gem-miR-15a exhibited potent anti-proliferative activity with IC50 values in the low nanomolar range, achieving ∼100-5000-fold greater potency relative to 5-FU and oxaliplatin. Importantly, it retained efficacy in both 5-FU- and oxaliplatin-resistant CRC models, effectively overcoming acquired chemoresistance. Mechanistically, Gem-miR-15a induced S-phase cell cycle arrest, eliminated the G2-phase cell population, and triggered apoptosis, accompanied by suppression of key oncogenic targets including WEE1, CHK1, YAP1 and BMI1. RNA-seq analysis further demonstrated modulation of pathways such as p53 signaling and reversal of resistance-associated gene expression, that were corroborated at the protein level. In vivo, Gem-miR-15a significantly reduced tumor growth at a dose ∼12-fold lower than gemcitabine, with no observable toxicity.

Gem-miR-15a represents a potent, multi-targeted therapeutic strategy capable of overcoming chemoresistance in CRC. Its enhanced stability, effective delivery and robust efficacy across resistant models and a favorable safety profile highlight its strong potential for clinical translation. Competing Interest Statement The authors have declared no competing interest. List of abbreviations - Gem-miR-15a - gemcitabine modified miRNA-15a - 5-FU - 5-fluorouracil - CRC - Colorectal cancer - miRNA - MicroRNA - miR-15a - MicroRNA-15a - Gem - Gemcitabine - PDAC - Pancreatic ductal adenocarcinoma