Background The progression of colorectal cancer (CRC) occurs via the PI3K/AKT/mTOR signaling pathway. Recent evidence suggests that microRNAs (miRNAs) can modulate gene expression, potentially offering new treatment options. This study aims to identify a miRNA that enhances radiosensitivity in CRC by targeting the PI3K/AKT/mTOR signaling pathway. Methods Gene expression datasets of CRC patients were retrieved from the NCBI database. Genes from the PI3K/AKT/mTOR signaling pathway were identified using the KEGG database. miRNA-mRNA binding sites were determined using TarBase, miRTarBase, mirDIP and miRNet. The selected miRNA was transfected into CRC cell lines, focusing on the two most responsive lines. Radiosensitivity was assessed by clonogenic assays and analysis of apoptotic cells in both irradiated and non-irradiated groups. Changes in gene expression were analyzed by real-time PCR. Results Data from 87 CRC patients were extracted and revealed 20 genes associated with the pathway according to specific criteria (logFC > 0 and adj.P < 0.05).miR-16-5p was selected for further investigation. The optimal transfection time was set at 48 h, with LoVo and HT-29 selected as cell lines. Irradiation with 4 Gy significantly suppressed miR-16-5p in both lines (P < 0.0023 for LoVo; P < 0.0001 for HT-29). miR-16-5p effectively suppressed the target genes CCNE1, CCND1, MYC, CDK4, HSP90AB1 and PIK3CA, with remarkable changes in gene expression after irradiation. Transfection and irradiation decreased cell survival (P < 0.05) and increased apoptosis (P < 0.0001). Conclusion The PI3K/AKT/mTOR signalling pathway is associated with radioresistance in CRC. miR-16-5p represses pathway genes and increases radiosensitivity, suggesting that miRNA-based gene therapy may improve treatment outcomes and patient quality of life.

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