Abstract PR12: Extracellular miR-92a secreted by human leukemia cells enhances endothelial cell migration in HUVECs

Abstract

Abstract Background and Aim: The miR-17-92 cluster has a potent tumor angiogenesis-regulating activity, and it appears to act as a cluster in which the individual miRNAs show intricate activity. Recently, the existence of extracellular miRNAs enclosed in exosomes has raised the possibility that they play an important role in cell-cell interaction. To elucidate whether or not extracellular miRNAs derived from neoplastic cells transfer into endothelial cells and become functionally active in the recipient cells, we investigated the interaction of a leukemia cell line (K562 cells) and human umbilical vein endothelial cells (HUVECs), because K562 cells release the miR-17-92 cluster, particularly miR-92a, into the extracellular environment. Experimental Procedures: K562 cells and HUVECs were cocultured separately using a Transwell filter (0.4-μm pore, Corning). After coculture for 24 h, the culture medium was collected and extracellular miRNAs were isolated using the mirVana PARIS kit (Ambion). The Pre-miR miRNA precursor (hsa-miR-92a; Ambion) was labeled using the Label IT siRNA Tracker Cy3 kit (Mirus), and K562 cells were transfected with 10 nM of the Cy3-labeled PremiR miRNA precursor. The day following transfection, cells were washed three times with PBS, and the culture medium was replaced with fresh serum-free AIM V medium (Invitrogen). After incubation for 24 h, the culture medium was collected, and the exosomal fraction was isolated using Exoquick (System Biosciences). Results: Transfer of extracellular miR-92a from the donor cells to the endothelial cells. K562 cells transfected with Cy3-labeled Pre-mir-92a were noncontact cocultured with HUVECs. Twenty-four hours after coculture, Cy3-miR-92a signals were detected in the cytoplasm of HUVECs. Moreover, Cy3-miR-92a colocalized with the signals of CD63, an exosomal marker. Exosomal miR-92a directly regulates the target gene. We performed a luciferase reporter assay and examined the expression of integrin α5, a target gene for miR-92a. Luciferase activity was markedly reduced and a significant suppression of integrin α5 expression by extracellular miR-92a was observed in HUVECs. This indicates that exogenous miRNA via exosomal transport can function equally as endogenous miRNA in HUVECs. Exosomal miR-92a enhances endothelial cell migration in HUVECs. We found that HUVECs incorporating exosomal miRNA showed active movement. In addition, wound-healing assay revealed that there were more Cy3-labeled miR-92a signals in migrating HUVECs, but the incorporation of extracellular miR-92a to HUVECs was inhibited by cytochalasin D, a migration inhibitor. This indicates that the incorporation of exosomal miRNA may be closely linked to the movement of HUVECs. Notably, cell migration assay using an 8-μm pore Transwell revealed that an excess of extracellular miR-92a strongly enhanced endothelial cell migration. Conclusions: Our study showed that extracellular miRNAs originating from neoplastic cells are incorporated into endothelial cells via exosomal transport and exert tumor-angiogenic function in endothelial cells. HUVEC migration enhanced the uptake of extracellular miR-92a, and an excess of extracellular miR-92a enhanced HUVEC migration. Our results provide a new insight into the angiogenic function of miR-92a, and revealed that extracellular miRNAs play an important role in neoplasia-to-endothelial cell communication. This abstract is also presented as Poster C25. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR12.