Characterization of miRNAs in Milk Small Extracellular Vesicles from Enzootic Bovine Leukosis Cattle

Abstract

Enzootic bovine leukosis (EBL) is a B-cell lymphosarcoma caused by the bovine leukemia virus (BLV). Most BLV-infected cattle show no clinical signs and only some develop EBL. The pathogenesis of EBL remains unclear and there are no methods for predicting EBL before its onset. Previously, it was reported that miRNA profiles in milk small extracellular vesicles (sEVs) were affected in cattle in the late stage of BLV infection. It raised a possibility that miRNA profile in milk sEVs from EBL cattle could be also affected. To characterize the difference in milk of EBL cattle and healthy cattle, we examined the miRNA profiles in milk sEVs from four EBL and BLV-uninfected cattle each using microarray analysis. Among the detected miRNAs, three miRNAs—bta-miR-1246, hsa-miR-1290, and hsa-miR-424-5p—which were detectable using quantitative real-time PCR (qPCR) and are associated with cancers in humans—were selected as biomarker candidates for EBL. To evaluate the utility of these miRNAs as biomarkers for EBL, their levels were measured using milk that was freshly collected from 13 EBL and seven BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p, but not hsa-miR-1290, were detected using qPCR and their levels in milk sEVs from EBL cattle were significantly higher than those in BLV-uninfected cattle. bta-miR-1246 and hsa-miR-424-5p in sEVs may promote metastasis by targeting tumor suppressor genes, resulting in increased amounts in milk sEVs in EBL cattle. These results suggest that bta-miR-1246 and hsa-miR-424-5p levels in milk sEVs could serve as biomarkers for EBL.

Identification of Suitable Internal Control miRNAs in Bovine Milk Small Extracellular Vesicles for Normalization in Quantitative Real-Time Polymerase Chain Reaction

“…To identify the candidate internal control miRNAs in bovine milk sEVs, we compared the microarray raw data published previously [ 6 , 7 ]. A total of 786 miRNAs in bovine milk sEVs were identified as common between the two datasets (data are not shown).…”

“…Based on the microarray data of the bovine milk sEVs obtained from our previous studies [ 6 , 7 ], five candidate internal control miRNAs in bovine milk sEVs ( n = 12) were quantified by the qPCR. All candidate internal control miRNAs were detected in the bovine milk sEVs using a qPCR.…”

“…To select the candidate internal control miRNAs in bovine milk sEVs, previously published microarray raw data were compared and evaluated [ 6 , 7 ]. To identify the common miRNAs in the two datasets, the raw data of miRNAs were combined to generate a single dataset.…”

“…Previous studies have reported that alterations in miRNA expression are useful for understanding the pathophysiological status of hosts [ 4 , 5 ]. For example, hsa-miR-424-5p in bovine milk sEVs are useful as biomarkers for the identification of cattle with a high risk of bovine leukemia virus (BLV) transmission and enzootic bovine leukosis (EBL), respectively [ 6 , 7 ]. Thus, miRNAs play important roles in defining both normal and pathologically specific gene expression patterns.…”

“…However, the commercial kits available for miRNA extraction from bovine milk sEVs are limited. Previously, we identified miRNA biomarkers in milk sEVs for cattle with a high risk of BLV transmission and EBL cattle [ 6 , 7 ]. However, there has been a lack of identification and use of an internal control miRNA in bovine milk sEVs for normalization in a quantitative real-time polymerase chain reaction (qPCR).…”