Methodological Approaches to Study Extracellular Vesicle miRNAs in Epstein⁻Barr Virus-Associated Cancers

Abstract

Epstein Barr-virus (EBV) was the first virus identified to be associated with human cancer in 1964 and is found ubiquitously throughout the world's population. It is now established that EBV contributes to the development and progression of multiple human cancers of both lymphoid and epithelial cell origins. EBV encoded miRNAs play an important role in tumor proliferation, angiogenesis, immune escape, tissue invasion, and metastasis. Recently, EBV miRNAs have been found to be released from infected cancer cells in extracellular vesicles (EVs) and regulate gene expression in neighboring uninfected cells present in the tumor microenvironment and possibly at distal sites. As EVs are abundant in many biological fluids, the viral and cellular miRNAs present within EBV-modified EVs may serve as noninvasion markers for cancer diagnosis and prognosis. In this review, we discuss recent advances in EV isolation and miRNA detection, and provide a complete workflow for EV purification from plasma and deep-sequencing for biomarker discovery.

Keywords: exosomes; extracellular vesicle; herpesvirus; microRNA; microvesicles; oncosomes.

Figure 1

Workflow of miRNAs profiling from blood extracellular vesicles (EVs). Plasma or serum samples were pretreated with thrombin and RNase A to remove fibrin and non-EV RNA contaminate. 1 mL of cleared plasma was loaded on size-exclusion chromatography (SEC) column and 1 mL fraction from 3 to 6 were collected and pooled. Concentrated EVs samples were quantified by nanoparticle tracking analysis (NTA), and 50% to 80% recovery could be achieved. Total EV RNAs were isolated by Qiagen miRNeasy micro kit. After checking total RNA with pico chip on a bioanalyzer, small RNA libraries were prepared by using NEBNext Small RNA Library Prep Set and run on an Illumina sequencer.