Exploration of microRNA Biomarkers in Blood Small Extracellular Vesicles for Enzootic Bovine Leukosis

Abstract

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). While most infected cattle show no clinical signs, approximately 30% of infected cattle develop persistent lymphocytosis (PL), and a small percentage may develop EBL. Currently, there is no method for predicting the possibility of EBL onset. In this study, we analyzed the microRNAs (miRNAs) encapsulated in small extracellular vesicles (sEVs) in the blood to explore the biomarkers of EBL. To identify candidate biomarkers, blood samples were collected from three BLV-uninfected and three EBL cattle. Total RNA was extracted from filtered serum and used for microarray analysis. Due to their association with cancer in human orthologs, we selected three miRNAs as candidate biomarkers, bta-miR-17-5p, bta-miR-24-3p, and bta-miR-210, which were more than twice as abundant in EBL cattle than in BLV-uninfected cattle. Quantitative real-time polymerase chain reaction (qPCR) using serum RNAs from six cattle used for the microarray analysis was carried out for the detection of the three selected miRNAs. Additionally, bta-miR-92a, whose ortholog has been associated with cancer in humans, was also examined by qPCR. bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a, were successfully detected, but bta-miR-210 was not. To further evaluate the utility of these three miRNAs as biomarkers, new blood samples were collected from 31 BLV-uninfected and 30 EBL cattle. The levels of bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a, were significantly higher in EBL cattle than in BLV-uninfected cattle. These results suggest that increased levels of bta-miR-17-5p, bta-miR-24-3p, and bta-miR-92a in the blood could be used as biomarkers for EBL. This study may contribute to the control of BLV infections and develop a prediction method of EBL onset.