Evidence for a TMAO-HULC-miRNA regulatory axis in colorectal cancer cells

Colorectal cancer (CRC) is a malignancy with a significant global disease burden.Trimethylamine N-oxide (TMAO), a gut microbiota-derived metabolite, has been implicated in tumorigenesis. The oncogenic long non-coding RNA highly upregulated in liver cancer (HULC) plays a pivotal role in CRC progression. However, the exact molecular mechanism of HULC and its correlation with TMAO in CRC pathogenesis has remained unclear. This study tested whether TMAO regulates HULC and whether HULC mediates changes in selected miRNAs relevant to CRC. Caco-2 cells were treated with TMAO (300 µM, 24 h) and HULC expression was quantified by RT-qPCR. HULC was transiently silenced using CRISPR/Cas13 (CasRx) and the expression of candidate downstream miRNAs (miR-21-5p, miR-200a-3p and miR-34a-5p) was measured by stem-loop RT-qPCR. Data are presented as mean ± SD of at least three independent biological replicates. Group differences were analyzed by ANOVA with appropriate post-hoc testing. TMAO treatment significantly increased HULC expression in Caco-2 cells. TMAO also elevated miR-21-5p and miR-200a-3p levels; these increases were attenuated when HULC was silenced. miR-34a-5p expression was not significantly affected by TMAO or by HULC knockdown. This study demonstrates that TMAO upregulates the oncogenic lncRNA HULC, and this upregulation is associated with increases the expression of miR-21-5p and miR-200a-3p. These findings reveal a TMAO-HULC signaling axis that positively influences the levels of oncogenic miRNAs. However, since a single cell line model was used in this study, it needs for further investigation across diverse CRC cell lines to confirm its generalizability.