5' isoforms of miR-1246 have distinct targets and stronger functions compared to canonical miR-1246 in colorectal cancer cells in vitro

Due to alternative processing pathway and post-transcriptional modifications, precursor miRNAs maturate into various sequence isoforms (isomiRs). These sequence variations may result in the changes of the miRNA seed site, target genes, involvement in signaling pathways and thus function. It is important to mention that knowledge about the targets of isomiR is still poor. To date, isomiR research has only been performed in melanoma, breast, and gastric cancer, but there are no experimental studies conducted in colorectal cancer. Here, we aimed to evaluate the putative targets and functional role in vitro of miR-1246 and its two 5' isoforms (ISO-miR-1246_a and ISO-miR-1246_G). To our best knowledge, this is the first study showing the important role of 5'isoforms of miR-1246 in colorectal carcinogenesis, while acting on different targetomes and being involved in distinct signaling pathways. Overall design: Colorectal cancer (CRC) is a multifactorial disease involving genetic and epigenetic factors, such as miRNAs. Sequencing-based studies revealed that miRNAs have many isoforms (isomiRs) with modifications at 3', 5' ends or in the middle, resulting in distinct targetomes, and consequentially, function. In the present study, we aimed to evaluate the putative targets and functional role in vitro of miR-1246 and its two 5' isoforms (ISO-miR-1246_a and ISO-miR-1246_G). Commercial Caco-2, SW480 and HT-29 cells of CRC origin were analyzed for expression of WT-miR-1246 and its 5' isoforms using small RNA sequencing data and overabundance of the two miR-1246 isoforms was determined in all cell lines. Transcriptome analysis of Caco-2 cell transfected with WT-miR-1246, ISO-miR-1246_G and ISO-miR-1246_a indicated a minor overlap of targetomes between the studied miRNA isoforms. Consequentially, enrichment analysis showed involvement of the potential targets of miR-1246 isoforms in distinct signaling pathways. Cancer related pathways were predominantly more enriched in dysregulated genes in ISO-miR-1246_G and ISO-miR-1246_a, whereas cell cycle pathways - in WT-miR-1246. Functional analysis of WT-miR-1246 and its two 5' isoforms revealed that inhibition of any of these molecules had a tumor-suppressive role (reduced cell viability, migration, and promoted early cell apoptosis) in CRC cells. However, 5'-isoforms had a stronger effect on viability compared to WT-miR-1246. To conclude, this research shows that WT-miR-1246 and its two 5'-isoforms have different targetomes and are involved in distinct signaling pathways, but collectively play an important role in CRC pathogenesis.