Serum bta-miRNA-375 as a potential biomarker for the early diagnosis of enzootic bovine leukosis.

To identify a biomarker for the early diagnosis of enzootic bovine leukosis (EBL) caused by bovine leukemia virus (BLV), we investigated the expression of a microRNA, bta-miR-375, in cattle serum. Using quantitative reverse-transcriptase PCR analysis, we measured bta-miR-375 levels in 27 samples from cattle with EBL (EBL cattle), 45 samples from animals infected with BLV but showing no clinical signs (NS cattle), and 30 samples from cattle uninfected with BLV (BLV negative cattle). In this study, we also compared the kinetics of bta-miR-375 with those of the conventional biomarkers of proviral load (PVL), lactate dehydrogenase (LDH), and thymidine kinase (TK) from the no-clinical-sign phase until EBL onset in three BLV-infected Japanese black (JB) cattle. Bta-miR-375 expression was higher in NS cattle than in BLV negative cattle (P < 0.05) and greater in EBL cattle than in BLV negative and NS cattle (P < 0.0001 for both comparisons). Receiver operating characteristic curves demonstrated that bta-miR-375 levels distinguished EBL cattle from NS cattle with high sensitivity and specificity. In NS cattle, bta-miR-375 expression was increased as early as at 2 months before EBL onset-earlier than the expression of PVL, TK, or LDH isoenzymes 2 and 3. These results suggest that serum miR-375 is a promising biomarker for the early diagnosis of EBL.

Characterization of microRNA expression in B cells derived from Japanese black cattle naturally infected with bovine leukemia virus by deep sequencing.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a malignant B cell lymphoma. However, the mechanisms of BLV-associated lymphomagenesis remain poorly understood. Here, after deep sequencing, we performed comparative analyses of B cell microRNAs (miRNAs) in cattle infected with BLV and those without BLV. In BLV-infected cattle, BLV-derived miRNAs (blv-miRNAs) accounted for 38% of all miRNAs in B cells. Four of these blv-miRNAs (blv-miR-B1-5p, blv-miR-B2-5p, blv-miR-B4-3p, and blv-miR-B5-5p) had highly significant positive correlations with BLV proviral load (PVL). The read counts of 90 host-derived miRNAs (bta-miRNAs) were significantly down-regulated in BLV-infected cattle compared to those in uninfected cattle. Only bta-miR-375 had a positive correlation with PVL in BLV-infected cattle and was highly expressed in the B cell lymphoma tissue of EBL cattle. There were a few bta-miRNAs that correlated with BLV tax/rex gene expression; however, BLV AS1 expression had a significant negative correlation with many of the down-regulated bta-miRNAs that are important for tumor development and/or tumor suppression. These results suggest that BLV promotes lymphomagenesis via AS1 and blv-miRNAs, rather than tax/rex, by down-regulating the expression of bta-miRNAs that have a tumor-suppressing function, and this downregulation is linked to increased PVL.