Abstract
MicroRNAs (miRNAs) are a class of small non-coding, endogenous regulatory RNAs that function by controlling gene expression at the post-transcriptional level. Using small RNA sequencing and qRT-PCR techniques, we found that the expression of miR-152 was significantly increased during lactation in the mammary glands of dairy cows producing high quality milk compared with that in cows producing low quality milk. Furthermore, DNA methyltransferase 1 (DNMT1), which is a target of miR-152, was inversely correlated with the expression levels of miR-152 in the mammary glands of dairy cows. Dairy cow mammary epithelial cells (DCMECs) were used as in vitro cell models to study the function of miR-152. The forced expression of miR-152 in DCMECs resulted in a marked reduction of DNMT1 at both mRNA and protein levels. This in turn led to a decrease in global DNA methylation and increased the expression of two lactation-related genes, serine/threonine protein kinase Akt (Akt) and peroxisome proliferator-activated receptor gamma (Pparγ). In contrast, inhibition of miR-152 showed the opposite results. By using an electronic Coulter counter (CASY-TT) and flow cytometer, we discovered that miR-152 enhanced the viability and multiplication capacity of DCMECs. In conclusion, miR-152 plays an important role in the development and lactation processes in the mammary glands of dairy cows. Our data provide insights into dairy cow mammary gland development and lactation.
Highlights
Using small RNA sequencing, we found that the level of miR-152 was significantly up-regulated in the mammary gland tissues of cows producing high quality milk (H) compared with that in cows producing low quality milk (L) as shown in Figure 1A (P,0.01)
We observed that the expression of miR-152 was markedly higher in the mammary gland tissues of cows producing H compared with those producing L using qRT-PCR, validating the small RNA sequencing results (Figure 1B, P,0.05)
To determine the levels of maintenance methylation in dairy cow mammary gland tissues, we measured the levels of DNA methyltransferase 1 (DNMT1) mRNA using qRT-PCR and found that DNMT1 was significantly down-regulated in the mammary gland tissues of cows producing H compared with that in cows producing L (Figure 2A, P,0.05)
Summary
MicroRNAs (miRNAs) are endogenous, non-coding, functional RNAs. Mature miRNA is a double-stranded RNA that contains approximately 22 nucleotides long generated from pre-miRNAs by the mediator of the RNAse III family member called Dicer [1]. MiR-152 is one of the three members of the miR-148/152 family, which contain miR-148a, miR-148b and miR-152. Recent studies have demonstrated that the aberrant expression of miRNAs in mammary glands may have crucial effects on mammary gland development and lactation [6,7,8,9]. We found that the expression of miR-152 differed in cow mammary gland tissues during the various lactation periods using small RNA sequencing and qRT-PCR. Therfore, it was proposed that miR-152 could regulate mammary gland development and lactation at the post-transcriptional level in Holstein dairy cows
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