Extracellular Vesicle-Mediated Transfer of a Novel Long Noncoding RNA TUC339: A Mechanism of Intercellular Signaling in Human Hepatocellular Cancer

Abstract

Although the expression of long noncoding RNA (lncRNA) is altered in hepatocellular cancer (HCC), their biological effects are poorly defined. We have identified lncRNA with highly conserved sequences, ultraconserved lncRNA (ucRNA) that are transcribed and altered in expression in HCC. Extracellular vesicles, such as exosomes and microvesicles, are released from tumor cells and can transfer biologically active proteins and RNA across cells. We sought to identify the role of vesicle-mediated transfer of ucRNA as a mechanism by which these novel lncRNA could influence intercellular signaling with potential for environmental modulation of tumor cell behavior. HCC-derived extracellular vesicles could be isolated from cells in culture and taken up by adjacent cells. The expression of several ucRNA was dramatically altered within extracellular vesicles compared to that in donor cells. The most highly significantly expressed ucRNA in HCC cell-derived extracellular vesicles was cloned and identified as a 1,198-bp ucRNA, termed TUC339. TUC339 was functionally implicated in modulating tumor cell growth and adhesion. Suppression of TUC339 by siRNA reduced HCC cell proliferation, clonogenic growth, and growth in soft agar. Thus, intercellular transfer of TUC339 represents a unique signaling mechanism by which tumor cells can promote HCC growth and spread. These findings expand the potential roles of ucRNA in HCC, support the existence of selective mechanisms for lncRNA export from cells, and implicate extracellular vesicle-mediated transfer of lncRNA as a mechanism by which tumor cells can modulate their local cellular environment. Intercellular transfer of functionally active RNA molecules by extracellular vesicles provides a mechanism that enables cells to exert genetic influences on other cells within the microenvironment.

Modulation of hypoxia-signaling pathways by extracellular long non-coding RNA regulator of reprogramming

“…We recently reported that extracellular vesicles derived from malignant hepatocytes contain microRNAs that can be transferred to other recipient cells and, moreover, can modulate cell signaling and cell viability in these recipient cells (Kogure et al, 2011). We have also identified lncRNAs, such as TUC339, that can be selectively enriched within these vesicles (Kogure et al, 2013). These observations support the presence of highly selective mechanisms for incorporating ncRNA into extracellular vesicles and support a role for ncRNAs as effectors of intercellular signaling (Valadi et al, 2007;Saunderson et al, 2008;Skog et al, 2008).…”

“…Human HCC cell lines HepG2, Hep3B, PLC-PRF5 and Huh-7 were obtained from the American Type Culture Collection (Manassas, VA), and HepG2.ST was derived from HepG2 cells by spontaneous transformation (Kogure et al, 2013). Cells were cultured in Dulbecco's modified Eagle's high-glucose medium (HyClone, Logan, UT), containing 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (Life Technologies, Grand Island, NY), at 37˚C under 5% CO 2 .…”

Trophic Effects of Mesenchymal Stem Cells in Tissue Regeneration

“…Subsets of mRNAs, miRNAs, and long noncoding RNAs can be transferred through EVs to recipient cells, inducing functional and phenotypic changes. 130,131 In particular, the horizontal transfer of miRNAs has been proposed as a new form of intercellular communication, representing a means by which donor cells can regulate the gene expression of recipient cells. 132 EVs extend the trophic repertoire of MSCs from the classical secreted growth factors to now include as well mRNAs, miRNAs, lipids, and membrane bound proteins.…”

Exosome-based liquid biopsy in the management of hepatocellular carcinoma

“…Kogure et al [55] first demonstrated that lncRNA with highly conserved sequences ultraconserved RNAs (ucRNAs) influences intercellular signalling. In HCC cell lines PLC/PRF/5 and Hep3B, the intercellular transfer of ucRNA TUC339 by exosomes represents a unique signalling mechanism by which tumour cells can promote HCC growth and spread [55] . Thus, the use of exosomes as biological delivery vehicles is of considerable interest.…”