N6-methyladenosine upregulates miR-181d-5p in exosomes derived from cancer-associated fibroblasts to inhibit 5-FU sensitivity by targeting NCALD in colorectal cancer

Resistance to 5-Fluorouracil (5-FU) is a frequent occurrence in patients with colorectal cancer (CRC). MicroRNAs (miRNAs) from cancer-associated fibroblasts (CAFs)-secreted exosomes have been associated with 5-FU sensitivity. The potential molecular mechanism of CAFs-exosomal miRNAs in CRC remains unclear. The aim of the present study was to elucidate the role of exosomal miRNAs in 5-FU sensitivity in CRC. Exosomes derived from CAFs were extracted. Exosomal miR-181d-5p was identified as a miRNA associated with 5-FU sensitivity. The putative function of exosomal miR-181d-5p was evaluated by ethynyl-2-deoxyuridine staining, flow cytometry, RNA immunoprecipitation, luciferase reporter assay, tumor xenograft formation, reverse transcription-quantitative PCR and western blot analysis. Modification of miR-181d-5p by the RNA N6-methyladenosine (m(6)A) methyltransferase like (METTL)3 was examined by m(6)A methylation analysis. The results indicated that m(6)A modification and METTL3 expression were upregulated in CRC patients. METTL3-dependent m(6)A methylation promoted the miR-181b-5p process by DiGeorge Syndrome Critical Region 8 (DGCR8) in CAFs. CAFs-derived exosomes inhibited 5-FU sensitivity in CRC cells through the METTL3/miR-181d-5p axis. A mechanistic study revealed that miR-181d-5p directly targeted neurocalcin δ (NCALD) to inhibit the 5-FU sensitivity of CRC cells. Patients with higher NCALD levels exhibited a higher survival rate. Taken together, METTL3-dependent m(6)A methylation was upregulated in CRC to promote the processing of miR-181d-5p by DGCR8. This led to increased miR-181d-5p expression, which inhibited the 5-FU sensitivity of CRC cells by targeting NCALD. The results of the present study provided novel insight into exosomal microRNAs in 5-FU sensitivity in CRC cells. Furthermore, exosomal miR-181d-5p may represent a potential prognostic marker for CRC.