Expression Profiles Of MicroRNAs From Lactating And Non-lactating Bovine Mammar Glands And Identification Of MiRNA Related To Lactation

MicroRNAs (miRNAs) are small non-coding RNA molecules that are approximately22nucleotides (nt) in length, which regulate specific target genes by mRNA degradation or translational repression. However, only a few studies have assessed the potential function of miRNAs in mammary gland lactogenesis. In this study, Solexa sequencing technology was used to obtain miRNA expression profiles from mammary gland tissue samples and microarray was performed to compare the difference in miRNA expression between periods of lactation (L) and non-lactation (NL). Using computational prediction, potential targets for these miRNAs were identified, leading to the construction of an interaction network related to lactation. Finally, the impacts of miR-484on glucose metabolism via HK23’UTR was studied. The results obtained are as follows:1Solexa sequencing of miRNA in bovine mammary glandTwo miRNA libraries were constructed using small RNA isolated from bovine mammary gland and sequenced using Solexa sequencing technology. A total of15,089.573and18,079,366reads were obtained from the L and NL period libraries, respectively. By filtering reference sequences,11,964,909and15,968,116clean reads were remained finally. Length distribution analysis revealed that more than half of these clean reads (52.6%) were22nt in length.2Features of miRNA in bovine mammary glandsA total of885pre-miRNAs encoding921miRNAs, of which884were unique, were detected with all clean reads. The unique miRNAs were categorized into three groups based on their hits:283known miRNAs,96conserved miRNAs and505novel candidates. The chromosomal positions of885pre-miRNAs were also studied. It was determined that800of the pre-miRNAs matched with the bta genome and230pre-miRNAs were grouped into55clusters.3Identification of differential expression patterns of miRNA in bovine MG and their target predictMicroarray and real time PCR were used to identify differentially expressed miRNAs on the basis of sequenced sequences. The expressions of69miRNAs were significantly different between L and NL periods (P<0.01, signal value>500). The283known miRNAs were predicted putative targets. More than10,000genes were selected as potential targets. All targets were then processed by GO analysis over half involved in transcriptional activity. Based on the results of target prediction, miRNAs and target gene interactions were integrated using EGAN to construct possible regulatory networks to investigate the relationship between miRNAs and lactation. The network includes37miRNAs detected by this study and15expressed target genes relating to lactation function.4Regulation of lactation genes by miRNAsThe HK2and STAT5genes and related miRNAs (miR-125b, miR-141, miR-181, miR-199a, miR-484and miR-500) in the network were selected to validate its reliability. The possible roles of these miRNAs in the regulation of gene expression were investigated using a cell-based model, RNAi, real-time PCR and western blotting tecnology. MiR-141mimics downregulated STAT5, while miR-141inhibitors increased STAT5protein levels. In order to examine the effect of these related miRNAs miRNAs on HK2expression, Mac-T cells were transfected with these related five miRNAs miRNA mimics. Transfection with miR-181a, miR-199b and miR-125b mimics did not significantly change the protein level of HK2(P>0.05), while miR-484and miR-500mimics markedly inhibited the production of HK2protein (P<0.05). Taken together with the fact that no significant differences were observed at the transcriptional level by real-time PCR, these results suggest that miR-484and miR-500may function at the translational level.5The impacts of miR-484on glucose metabolism and key enzymeSequence of bovine HK23’UTR was Firstly cloned using3’RACE. By the luciferase report system, ectopic expression of miR-484resulted in a significant reduction of reporter activity in Mac-T cells. It is confirmed that miR-484could regulate HK2expression via targeting the3’UTR of HK2. Additionally, we performed RNAi analyses of HK2with miRNA mimics. Overexpression of miR-484effectively reduced HK activity and glucose utilization.In summary, total884miRNAs were obtained from L and NL bovine mammary glands, and their category and chromosome postion were analysed. The expressions of69miRNAs were significantly different between L and NL periods. An interaction network of miRNAs and their target genes relating to lactation were constructed to postulate the functional roles of miRNAs in the mammary glands. It is validated that miR-484could reduce glucose utilization in cells via inhibiting HK activity by targeting the3’UTR of HK2. These results obtained from this study laid the foundation for understanding the signal pathway and scientific regulation in cows’ lactation.