通过新型吉西他滨修饰的miR-15a克服结直肠癌的耐药性

Colorectal cancer (CRC) is the third most prevalent cancer globally. Despite therapeutic advances, chemoresistance remains a major challenge, with ~20-30% of advanced-stage CRC patients experiencing recurrence within the first five years of treatment. Growing evidence suggests that targeting DNA damage response proteins, such as ATR/ATM, WEE1, and CHK1, is critical for overcoming resistance. In this regard, microRNAs (miRNAs) offer great therapeutic potential, as they can simultaneously target multiple signaling pathways and their dysregulation is widely implicated in cancer. Notably, tumor-suppressor miRNA-15a (miR-15a) is frequently downregulated in CRC and has been associated with poor patient prognosis. While miR-15a restoration has shown great promise, effective miRNA delivery remains a significant challenge due to its instability. Various chemical modifications have been shown to greatly enhance the stability of miRNAs. One such modification, 5-FU modified miR-15a (5-FU-miR-15a) was demonstrated as a promising therapeutic in CRC by our lab. Building on this, we engineered a new gemcitabine-modified miR-15a (Gem-miR-15a), which integrates the tumor-suppressive properties of miR-15a with the chemotherapeutic ability of gemcitabine. Though not a standard therapy in CRC, gemcitabine is emerging as an alternative for advanced refractory and resistant cases. Thus, we hypothesized that Gem-miR-15a would have an enhanced therapeutic advantage in overcoming chemoresistance to standard drugs, such as 5-FU. In this study, we assessed the effects of Gem-miR-15a on cell viability, apoptosis, and cell cycle progression of various parental (HCT116, SW480, SW620, HT-29) and 5-FU resistant CRC cell lines. Gem-miR-15a drastically reduced cell viability in both parental (IC 50 =1-10nM) and resistant cells (IC 50 =4.08 nM) without any delivery vehicle, significantly induced apoptosis, and caused an S-phase cell cycle arrest. Gem-miR-15a demonstrated dramatically increased cytotoxicity, with an almost 1000-fold reduction in IC 50 as compared to the standard drug, 5-FU. The effects were consistent in 3D spheroids (IC 50 =9.76 nM) and patient-derived organoids (IC 50 =6-14 nM), too. The modification retained the target specificity of the native miR-15a, and downregulated key oncogenes like WEE1, CHK1 and BMI1 which was confirmed by western blotting. Additionally, Gem-miR-15a demonstrated strong synergy with oxaliplatin (Synergy score: 11.03). Experiments were done in triplicate (n=3) and analyzed using Student's t-test (p < 0.05). Gem-miR-15a (4mg/kg) was also significantly able to reduce the tumor growth in in vivo metastatic mouse models, as compared to the mice treated with vehicle alone, with no visible toxicities. Hence, our findings establish Gem-miR-15a as a potent and multi-targeted therapeutic candidate capable of overcoming chemoresistance in CRC.