This is the first report describing the analysis of circulating miRNA profiles in Japanese Black cattle during early gestation. Although circulating miRNAs have already been reported in pregnant Holstein-Friesian cows [13–16], the application of these miRNAs in Japanese Black cows remains unclear because breed-specific miRNA expression has been observed in some cattle breeds [17, 18]. Furthermore, it is necessary to clarify another miRNA candidate for early pregnancy diagnosis in cows and to investigate their potential for diagnosis. In the present study, we demonstrated that miR-19b, miR-25, miR-29a, and miR-148a levels in plasma were elevated in pregnant Japanese Black cows on day 21 after AI compared with the non-pregnant group. These circulating miRNA levels did not change during the estrous cycle in the early to late stages and were not affected by the degree of hemolyzed plasma.
Recently, remarkable advances in RNA-sequencing (RNA-seq) technology in gene expression analysis have also been applied to the expression analysis of miRNAs as small RNA-seq [22]. Many of the previously reported analyses of circulating miRNAs in cows have also used RNA-seq [1–3]. Oligonucleotide DNA microarray has been used for comprehensive analysis of gene expression, and we have also studied pregnancy diagnosis using this microarray to measure gene expression in bovine peripheral blood leukocytes [10]. In oligonucleotide DNA microarray, probes to be placed on array slides can be custom-designed, which has the advantage of not requiring the complicated analysis techniques necessary for RNA-sEq. Here, we attempted a global expression analysis of bovine circulating miRNAs using oligonucleotide DNA microarray from the accumulation of our knowledge and techniques. In the present study, the microarray detected 124 miRNAs in bovine plasma, which is less than that of approximately 300 to 800 miRNAs in bovine plasma or serum, as previously reported [13–16]. This could have been inferior to RNA-seq in sensitivity detection using oligonucleotide DNA microarray. However, miR-19b [14], miR-25 [15], miR-26b [13, 14, 16], miR-27b [16], miR-29a [15], and miR-148a [16], which showed high values in the pregnant herds extracted from our microarray experiments, tended to be similar to the results of the previous RNA-seq or PCR array analysis. Therefore, these reports support the microarray results of the present study.
Appropriate normalization using reference genes is crucial for performing RT-qPCR and accurate miRNA expression analysis [23]. The selection of erroneous reference genes greatly influences the quantitative results [24, 25]. NormFinder is a software used to assess gene expression stability and is useful for selecting endogenous controls [19]. Therefore, we searched for reference miRNAs in the plasma of Japanese Black cattle for more precise miRNA determination. In the present study, 11 reference miRNA candidates, including two reference miRNAs used in previous reports [14, 20], were validated by RT-qPCR. Validation experiments showed that miR-2348 and miR-3141 could not be amplified specifically by RT-qPCR with the prepared primer set in the present study and could not be accurately quantified. Comparing the Ct-values between the non-pregnant and pregnant groups for each miRNA, significant differences were observed in let-7g, miR-128, miR-2478, and miR-2888. These miRNAs may, however, be unsuitable as reference miRNAs for comparing miRNA expression in non-pregnant and pregnant Japanese Black cows. NormFinder analysis recommended the use of miR-2455 as the optimal reference miRNA. In addition, the reference miRNA candidates, except miR-2888, in the present study were more stable than the previously described candidates let-7g and miR-128. Therefore, the reference miRNA candidates selected in this study may be considered superior to previously reported reference genes. For the quantification of miRNA by RT-qPCR, standardization using the measurement results of the reference gene is critical, but no particular reference gene exists that can be applied to all tissues or plasma. [26]. Thus, the selection of appropriate reference genes requires validation between specific populations for comparison and types of samples such as tissues and plasma, and this study found appropriate reference genes for comparison between non-pregnant and pregnant groups using plasma. Although we have only validated the NormFinder algorithm for reference miRNA selection, RT-qPCR results showed that miR-2455 did not fluctuate between non-pregnant and pregnant females, making it reasonable to use miR-2455 as a reference miRNA.
The present results indicate that miR-19b, miR-25, miR-29a, and miR-148a may be useful indicators of pregnancy diagnosis in Japanese Black cows. Ioannidis and Donadeu (2017) reported the circulating miRNA profile of Holstein-Friesian heifers at days 0 and 60 post-AI using small RNA-seq and with RT-qPCR validation [14]. Although circulating miR-26b was only increased in pregnant cows validated by RT-qPCR, RNA-seq analysis showed elevated miR-19b levels on day 60 of pregnancy. Gebremedhn et al. (2018) reported 23 differentially expressed miRNAs, including miR-25 and − 29a, in serum on day 24 of pregnancy in Holstein-Friesian cows [15]. In another report, the RNA-seq approach revealed that miR-148a was increased on day 30 of pregnancy [16]. Although we could not find new miRNAs for early pregnancy diagnosis as an indicator, it was confirmed that miR-19b, miR-25, miR-29a, and miR-148a were indicators for pregnancy diagnosis in Japanese Black cows. However, the sample size was small for both non-pregnant and pregnant cows in this study. Therefore, larger studies are needed to determine the proportion of non-pregnant or pregnant cows that can be identified using this technique.
Cows have their first estrus around 10 months of age and then repeat their estrus with a cycle of approximately 21 days. Hormones such as estradiol, progesterone, and luteinizing hormone are involved in the development of estrus, and cyclic variations in blood levels of these hormones have also been observed [27]. Circulating miRNAs have also been reported to change during the estrous cycle [28], and miRNAs used in pregnancy diagnosis should be unaffected by the estrous cycle. Therefore, four miRNAs that have been biomarker candidates for pregnancy diagnosis in the present study were examined for changes in plasma from days 0 to 20 of the estrous cycle. No variation related to the estrous cycle was observed for any miRNA, indicating that these miRNAs were not affected by ovarian and pituitary hormones.
Circulating miRNAs are present in vesicular structures, such as exosomes, and are known to be relatively stable [29, 30]. Since blood can be collected relatively minimally invasively, there have been numerous studies aimed at using circulating miRNAs as biomarkers for various diseases. However, hemolysis may occur at the time of collection [30] and has been reported to affect plasma miRNA expression levels [30–32]. Therefore, in this study, the extent of hemolysis in miR-19b, miR-25, miR-29a, and miR-148a was determined. Hemolytic samples were quantified from free hemoglobin with 414 nm absorbance measurements and miR-23a and miR-451 content [21, 31]. The free hemoglobin and ratio of miR-451 to miR-23a in plasma increased with increasing hemolysis, and both showed significant differences compared with non-hemolyzed samples at 0.8% hemolysis or higher. The plasma appearance was also determined to be severe hemolysis, with hemolysis visible at 0.8% hemolysis or higher (pictures not shown). RT-qPCR showed that the content of miR-29a did not change significantly with hemolysis. Although miR-19b, miR-25, and miR-148a showed changes in content only in severely hemolyzed samples (4%), these miRNAs were largely unaffected by hemolysis, indicating that they may be useful as biomarker candidates in early pregnancy diagnosis.