Circulating RNAs as biomarkers in Epstein-Barr virus-associated nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is an aggressive tumour associated with Epstein-Barr virus (EBV), which has a poor prognosis when detected late. Easy methods for detecting NPC at earlier stages are highly needed. Host and viral miRNAs are perturbed in NPC, a fraction of which are included in extracellular vesicles and/or in ribonucleoprotein complexes that enter into the circulation. Measuring circulating miRNAs may provide a robust and direct insight of the oncogenic process. Here we analysed whether host and viral circulating miRNA fingerprints may reflect tumour burden and behaviour. Defined circulating microRNAs were analysed from total serum and plasma (NPC, non-NPC patients and healthy controls). Selected NPC sera and plasma were fractionated by size-exclusion chromatography into extracellular vesicle and lipoprotein fractions. Exosome-like vesicles released from EBV-infected NPC were characterized by western blot and transmission electron microscopy. Deep RNA sequencing (RNAseq) was done for NPC cell lines, exosomes and selected sera. EBV DNA load was measured as well. Viral miRNA (mirBART7, 9, 13), host miRNA (miR16, 21, 155) and small RNAs (EBER1 and vaultRNA) were detected by stemloop reverse transcription-polymerase chain reaction (RT-PCR). We detected viral miRNAs in NPC sera and plasma which levels seem to increase with TNM stage despite absence of detectable EBV DNA in some. EBER1 RNA was absent in virtually all NPC sera and plasma, despite high levels in NPC tissues. Cellular miR155, which expression is induced by EBV-LMP1, was significantly elevated in NPC when compared to controls. Fractionation from NPC sera and plasma revealed that miR155 and vault RNA are selectively enriched in extracellular vesicles, whereas viral miRNAs are more randomly distributed. The pattern of tetraspanin (CD63, CD81) protein expression is heterogeneous in NPC patients compared to healthy controls. Further, RNAseq and RT-PCR analysis are being performed to determine the diagnostic potential of circulating miRNAs for NPC.