miRNA-sequencing and bioinformatics analyses of key miRNAs and their associated genes regulated by EC-synthetic retinoids treatment in Caco-2 cancer cells

EC-synthetic retinoids are derivatives of vitamin A with diverse biological activities, including anti-cancer activity, but their potential impact on miRNA regulation is still unclear. Therefore, the aim of the current study is to explore the effect of two EC-synthetic retinoids (EC19 and EC23) compared to the parent natural all-transretinoic acid (ATRA) on the differential expression of miRNA profiles using the previously reported sensitive Caco-2 cell line as an in-vitro model. High-throughput miRNA sequencing revealed the top 10 upregulated (fold change from zero to >1) and 10 downregulated miRNAs (fold change from zero to <1) in Caco-2 cells compared to WI-38 normal cells. All retinoid treatments showed that hsa-miR-6817-3p, hsa-miR-548f-3p, hsa-miR-1538, and hsa-miR-5690 were downregulated, and hence their expression pattern was reversed. ATRA was able to upregulate all 10 miRNAs, while EC19 did not show modulation of any of these miRNAs, and EC23 was able to upregulate only hsa-miR-548f-3p. The miRNAs hsa-miR-10,400-5p and hsa-miR-4747-5p were unique and downregulated after all retinoid treatments. Concerning other miRNAs, some were specifically downregulated after individual retinoid treatment, such as hsa-miR-4667-5p (ATRA and EC19), hsa-miR-6088, and hsa-miR4508 (EC-synthetic retinoids). Post-validation assessment using RT-qPCR on selected miRNAs after retinoid treatment showed that hsa-miR-548f-3p, hsa-miR-4508, and hsa-miR-6088 were consistent with high-throughput miRNA sequencing. KEGG pathway and miRNet analysis of retinoids-regulated miRNAs revealed some regulated genes involved in apoptosis, cell cycle, TGF-beta, p53, and carcinogenesis in colorectal cancer. The current data represent a solid basis for further functional studies to understand the mechanism of action of the EC-synthetic retinoids and other derivatives as well as potential oncomiRs or tumor suppressor miRNAs with diagnostic and prognostic value in colorectal cancer.