Extracellular vesicle-mediated miR-126-3p transfer contributes to inter-cellular communication in the liver tumor microenvironment

Extracellular vesicles (EVs) and their contents are gaining recognition as important mediators of intercellular communication through the transfer of bioactive molecules, such as non-coding RNA. The present study comprehensively assessed the microRNA (miRNA/miR) content within EVs released from HepG2 liver cancer (LC) cells and LX2 hepatic stellate cells (HSCs) and determined the contribution of EV miRNA to intercellular communication. Using both transwell and spheroid co-cultures of LC cells and HSCs, miR-126-3p within EV was established as a mediator of HSC to LC cell communication that influenced tumor cell migration and invasion, as well as the growth of multicellular LC/HSC spheroids. Manipulation of miR-126-3p either by enforced expression using pre-miR-126-3p or by inhibition using antimiR-126-3p did not alter tumor cell viability, proliferation or sensitivity to either sorafenib or regorafenib. By contrast, enforced expression of miR-126-3p decreased tumor-cell migration. Knockdown of miR-126-3p in tumor cells increased disintegrin and metalloproteinase domain-containing protein 9 (ADAM9) expression and in HSCs increased collagen-1A1 accumulation with an increase in compactness of multicellular spheroids. Within LC/HSC spheroids, ADAM9 and vascular endothelial growth factor expression was increased by silencing of miR-126-3p but diminished with the restoration of miR-126-3p. These studies implicate miR-126-3p in functional effects on migration, invasion and spheroid growth of tumor cells in the presence of HSCs, and thereby demonstrate functional EV-RNA-based intercellular signaling between HSCs and LC cells that is directly relevant to tumor-cell behavior.