In silico and in vitro analysis of the impact of single substitutions within EXO-motifs on Hsa-MiR-1246 intercellular transfer in breast cancer cell

Abstract

MiR-1246 has recently gained much attention and many studies have shown its oncogenic role in colorectal, breast, lung, and ovarian cancers. However, miR-1246 processing, stability, and mechanisms directing miR-1246 into neighbor cells remain still unclear. In this study, we aimed to determine the role of single-nucleotide substitutions within short exosome sorting motifs — so-called EXO-motifs: GGAG and GCAG present in miR-1246 sequence on its intracellular stability and extracellular transfer. We applied in silico methods such as 2D and 3D structure analysis and modeling of protein interactions. We also performed in vitro validation through the transfection of fluorescently labeled miRNA to MDA-MB-231 cells, which we analyzed by flow cytometry and fluorescent microscopy. Our results suggest that nucleotides alterations that disturbed miR-1246 EXO-motifs were able to modulate miRNA-1246 stability and its transfer level to the neighboring cells, suggesting that the molecular mechanism of RNA stability and intercellular transfer can be closely related.

The DNA-repair protein APE1 participates with hnRNPA2B1 to motif-enriched and prognostic miRNA secretion

“…We then validated APE1-hnRNPA2B1 interplay in the regulation of miRNAs containing an EXO-motif (see Supplementary Figure S8 and Supplementary Tables 8 and 9). Comparing all the obtained results, we identified hsa-miR-1246 as: i) the only DEmiRNA, in all the cell lines we tested so far (12, 17), having EXO-motif occurrences in both the precursor and mature forms; ii) a possible interactor of RBPs, based on structural studies showing the presence of EXO-motifs in its sequence (54) and, iii) a highly-expressed ncRNA in EVs derived from many human cancer cell lines (55).…”

“…As an exhaustive target, we characterized the interaction of APE1 with miR-1246, already explored in the sorting process within EVs (54) and for its paracrine role in tumor progression of different cancer types (55). miR-1246 has been found to be oncogenic in lung, cervical, liver, colorectal, breast, pancreatic and ovarian cancers through mechanisms regulating GSK3β, JAK/STAT, PI3K/AKT, RAF/MEK/ERK, Wnt/β-catenin, THBS2/ MMP and NOTCH2 pathways.…”

“…As an exhaustive target, we characterized the interaction of APE1 with miR-1246, already explored in the sorting process within EVs (54) and for its paracrine role in tumor progression of different cancer types (55).…”

Engineered EVs designed to target diseases of the CNS

“…For example, RNA-binding proteins (hnRNPU and SYNCRIP) were identified in regulating sorting mechanism of miRNAs into EVs [ 108 , 109 ]. And these exosome sorting motifs have been found to be important in modulating miRNA stability and its intracellular transfer [ 110 ]. Recently, it has been suggested that the packaging of mRNA and lncRNA into EVs not only impacts intracellular messaging between cells or organs, but also plays a role in transforming the transcriptome of the source cell [ 111 ].…”

Cell cycle arrest and apoptotic studies of Terminalia chebula against MCF-7 breast cancer cell line: an in vitro and in silico approach

“…Cancer is one of the deadliest illnesses worldwide. The global disease burden in 2018 was estimated to be 18.1 million new malignant cases ( 1 ). In India, there were approximately 2.25 million recorded cases and over 784,000 deaths in the year 2020 as per the National Institute of Disease Avoidance and Research.…”