Comprehensive analysis of blood cells and plasma identifies tissue-specific miRNAs as potential novel circulating biomarkers in cattle

Abstract Background The potential of circulating miRNAs as biomarkers of tissue function, both in health and disease, has been extensively demonstrated in humans. In addition, circulating miRNA biomarkers offer significant potential towards improving the productivity of livestock species, however, such potential has been hampered by the absence of information on the nature and source of circulating miRNA populations in these species. In addition, many miRNAs originally proposed as robust biomarkers of a particular tissue or disease in humans have been later shown not to be tissue specific and thus to actually have limited biomarker utility. In this study, we comprehensively analysed miRNA profiles in plasma and cell fractions of blood from cattle with the aim to identify tissue-derived miRNAs which may be useful as biomarkers of tissue function in this important food animal species. Results Using small RNA sequencing, we identified 92 miRNAs with significantly higher expression in plasma compared to paired blood cell samples (n = 4 cows). Differences in miRNA levels between plasma and cell fractions were validated for eight out of 10 miRNAs using RT-qPCR (n = 10 cows). Among miRNAs found to be enriched in plasma, we confirmed miR-122 (liver), miR-133a (muscle) and miR-215 (intestine) to be tissue-enriched, as reported for other species. Profiling of additional miRNAs across different tissues identified the human homologue, miR-802, as highly enriched specifically in liver. Conclusions These results provide novel information on the source of bovine circulating miRNAs and could significantly facilitate the identification of production-relevant tissue biomarkers in livestock. In particular, miR-802, a circulating miRNA not previously identified in cattle, can reportedly regulate insulin sensitivity and lipid metabolism, and thus could potentially provide a specific biomarker of liver function, a key parameter in the context of post-partum negative energy balance in dairy cows

Circulating microRNA Profiles during the Bovine Oestrous Cycle

Up to 50% of ovulations go undetected in modern dairy herds due to attenuated oestrus behavior and a lack of high-accuracy methods for detection of fertile oestrus. This significantly reduces overall herd productivity and constitutes a high economic burden to the dairy industry. MicroRNAs (miRNAs) are ubiquitous regulators of gene expression during both health and disease and they have been shown to regulate different reproductive processes. Extracellular miRNAs are stable and can provide useful biomarkers of tissue function; changes in circulating miRNA profiles have been reported during menstrual cycles. This study sought to establish the potential of circulating miRNAs as biomarkers of oestrus in cattle. We collected plasma samples from 8 Holstein-Friesian heifers on days Days 0, 8 and 16 of an oestrous cycle and analysed small RNA populations on each Day using two independent high-throughput approaches, namely, Illumina sequencing (n = 24 samples) and Qiagen PCR arrays (n = 9 sample pools, 3-4 samples / pool). Subsequently, we used RT-qPCR (n = 24 samples) to validate the results of high-throughput analyses, as well as to establish the expression profiles of additional miRNAs previously reported to be differentially expressed during reproductive cycles. Overall, we identified four miRNAs (let-7f, miR-125b, miR-145 and miR-99a-5p), the plasma levels of which distinctly increased (up to 2.2-fold, P < 0.05) during oestrus (Day 0) relative to other stages of the cycle (Days 8 and 16). Moreover, we identified several hundred different isomiRs and established their relative abundance in bovine plasma. In summary, our results reveal the dynamic nature of plasma miRNAs during the oestrous cycle and provide evidence of the feasibility of using circulating miRNAs as biomarkers of reproductive function in livestock in the future

Circulating miRNA signatures of early pregnancy in cattle

BACKGROUND: Low fertility remains a leading cause of poor productivity in dairy cattle. In this context, there is significant interest in developing novel tools for accurate early diagnosis of pregnancy. MicroRNAs (miRNAs) are short RNA molecules which are critically involved in regulating gene expression during both health and disease. MiRNAs have been shown to regulate ovarian function, uterine receptivity, embryonic development and placental function. Circulating miRNAs can provide useful biomarkers of tissue function and disease; importantly, differential miRNA profiles have been linked to pregnancy and preeclampsia in humans. This study sought to establish the potential of circulating miRNAs as biomarkers of early pregnancy in cattle. RESULTS: We applied Illumina small-RNA sequencing to profile miRNAs in plasma samples collected from eight non-pregnant heifers on Days 0, 8 and 16 of the oestrous cycle and 11 heifers on Days 16 and 24 of pregnancy. We sequenced a total of 46 samples and generated 9.2 million miRNA reads per sample. There were no differences in miRNA read abundance between any of the pregnant and non-pregnant time-points (FDR > 0.1). As a complementary approach, we analysed sample pools (3–4 samples/pool) corresponding to Days 0, 8 and 16 of the oestrous cycle and Day 24 of pregnancy (n = 3 pools/group) using Qiagen PCR arrays. A total of 16 miRNAs were differentially expressed (FDR < 0.1) in plasma between pregnant and non-pregnant animals. RT-qPCR validation using the same plasma samples confirmed that miR-26a was differentially upregulated on Day 16 pregnant relative to non-pregnant heifers (1.7-fold; P = 0.043), whereas miR-1249 tended to be upregulated in Day 16 pregnant heifers (1.6-fold; P = 0.081). Further validation in an independent group of heifers confirmed an increase in plasma miR-26a levels during early pregnancy, which was significant only on Day 24 (2.0-fold; P = 0.027). CONCLUSIONS: Through genome-wide analyses we have successfully profiled plasma miRNA populations associated with early pregnancy in cattle. We have identified miR-26a as a potential circulating biomarker of early pregnancy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-016-2529-1) contains supplementary material, which is available to authorized users