Systemically Circulating Viral and Tumor-Derived MicroRNAs in KSHV-Associated Malignancies

Abstract

MicroRNAs (miRNAs) are stable, small non-coding RNAs that modulate many downstream target genes. Recently, circulating miRNAs have been detected in various body fluids and within exosomes, prompting their evaluation as candidate biomarkers of diseases, especially cancer. Kaposi's sarcoma (KS) is the most common AIDS-associated cancer and remains prevalent despite Highly Active Anti-Retroviral Therapy (HAART). KS is caused by KS-associated herpesvirus (KSHV), a gamma herpesvirus also associated with Primary Effusion Lymphoma (PEL). We sought to determine the host and viral circulating miRNAs in plasma, pleural fluid or serum from patients with the KSHV-associated malignancies KS and PEL and from two mouse models of KS. Both KSHV-encoded miRNAs and host miRNAs, including members of the miR-17–92 cluster, were detectable within patient exosomes and circulating miRNA profiles from KSHV mouse models. Further characterization revealed a subset of miRNAs that seemed to be preferentially incorporated into exosomes. Gene ontology analysis of signature exosomal miRNA targets revealed several signaling pathways that are known to be important in KSHV pathogenesis. Functional analysis of endothelial cells exposed to patient-derived exosomes demonstrated enhanced cell migration and IL-6 secretion. This suggests that exosomes derived from KSHV-associated malignancies are functional and contain a distinct subset of miRNAs. These could represent candidate biomarkers of disease and may contribute to the paracrine phenotypes that are a characteristic of KS.

Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

“…EVs were resuspended in proportional volumes of PBS (see methods). The tetraspanin molecules CD9 and CD63 [8,25,26,38,41], the ESCRT-I associated tumour suppressor gene 101 (TSG101) [19,20,25,42–44], and the late-endosome/lipid raft protein Flotillin-2 [7,45–47] were selected as markers. All were present in EV obtained by these three methods, but not the intracellular control Tubulin (Figure 2(a)).…”

“…The viral miRNAs are incorporated into CD63+ EV [7] and thus RNase resistant. Therefore, any KSHV-encoded miRNAs (miRK) can be used to track EV through any purification process.…”

“…Isolation of EV from human plasma can present several issues that must be addressed such as: contamination with virus particles, contamination with high levels of lipoprotein-associated with LDLs and HDLs, and contamination with coagulation factors. To separate EV from viral particles an large lipoproteins, subsequent steps such as affinity purification using antibody coated beads should be employed [7,11]. …”

Extracellular vesicles: novel vehicles in herpesvirus infection

“…There are few available models for KSHV, but in a mouse model of KSHV and in patients with KSHVassociated malignancies, exosomal miRNAs of the host and those encoded by KSHV, including the miR-17-92 cluster, were successfully detected. Furthermore, the characterization and analysis of exosomal miRNAs, their targets, and endothelial cells revealed that exosomes containing a wide variety of miRNAs play a functional biological role in signaling pathways linked to the pathogenesis of KSHV and its phenotypic characteristics (Chugh et al, 2013;Zhang et al, 2015a). Hence, distinct exosomal miRNAs are expected to represent a portion of the paracrine signaling mechanism, which is the hallmark of KSHV tumorigenesis.…”

Exosomes in Viral Disease

“…Exosomes produced by infected cells were demonstrated to carry both KSHV and host-derived miRNA. Exposure to patientderived exosomes increased cell migration and IL-6 production in recipient endothelial cells [125]. Additionally, like EBV, KSHV reprogrammed the cell profile of infected B cells [126].…”