Screening And Functional Investigation Of Mirnas Regulating Milk Fat Metabolism In Dairy Goat Mammary Gland

Goat milk has higher fat content than cow milk. Goat milk also contains greater amounts of short-chain fatty acids, medium-chain fatty acids, and unsaturated fatty acids which are considered good for human health. These characteristics of goat milk may be attributable to the mechanism of milk fat metabolism. In comparison to cow, no specific gene in goat has been identified. Furthermore, the gene’s function identified in goat and cow is the same according to the current reports. It has been hypothesized that the difference in milk fat content and composition between goat and cow are caused by the difference in gene expression and regulation in each levels (i.e., post-transcription, post-translation). MicroRNAs (miRNAs) are small, non-coding RNAs that have emerged as key post-transcriptional regulators of gene expression in nearly all molecular metabolisms. MiRNAs have been reported to be important regulators for lipid and fat metabolism both in adipose and liver tissues. Little is known about the biological roles of miRNAs in goat mammary gland. To investigate the function of miRNAs regulating milk fat metabolism in goat mammary gland, we choose miRNAs as an entry point and performed a large scale miRNAs sequencing to obtain the miRNA profile in lactating mammary gland of Xinong Saanen dairy goats. We characterized goat miRNAs, measured their expression in mammary gand during different phases, and screened miRNAs associated with milk fat synthesis. Then, we over-expressed the miRNAs in goat mammary gland epithelial cells (GMEC) by using recombinant adenovirus, and measured the indices of milk fat synthesis of the GMEC. We also identified the synergistic regulation among miRNAs in mammary gland. This study is helpful with understanding the mechanism of lactation and milk fatty acid metabolism, and will provide a new subject for improving the quality of goat milk and increasing beneficial content of fatty acid in milk. The main results of this study were showed as following:1. Solexa sequencing technique was used to profile all small RNAs in mammary gland of lactating goats. After filtering low quality data,22,084,321clean reads of small RNAs were obtained. Among these small RNAs, the number of22nucleotides (nt) small RNAs (33.4%of total small RNAs reads) is the most dominant fragments. Based on the results of sequence alignment, five categories small RNA (tRNAs, rRNAs, snoRNAs, snRNAs, and miRNAs) were identified in goat mammary gland, of which miRNAs (10,534,221reads) has the highest percentage composition accounted for47.7%of total small RNAs reads. Goat mammary gland contains1,143miRNAs including931conserved miRNAs and212novel miRNA candidates. The931conserved miRNAs were aligned with current annotated miRNAs in the miRBase (Release17.0). Results of sequence alignment showed:(1) goat and cow (575miRNAs) have the largest number of conserved miRNAs, at meanwhile, goat and cow (116miRNAs) also have the largest number of specific miRNAs which have been identified only in two species;(2) Among conserved miRNAs,335miRNAs containing391end variations were identified. Frequency of base reduction is higher than base addition.3’base variations happen more easily than5’;(3)109base substitutions containing63transitions and46transversions were found.2. PCR was used to identify the expression levels of the read top30miRNAs in mammary gland during mid-lactation and dry period.17differently expressed miRNAs were identified. PicTar and TargetScan softwares were used to predict miRNA targets. GO Term and KEGG softwares were used to enrich the miRNAs targets. The majority of target function of the17miRNAs is associated with processes of development, proliferation and fat synthesis. Among the17miRNAs and10known function (regulating lipid metabolism in other mammals) miRNAs,4miRNAs (miR-23a, miR-27a, miR-103, and miR-200a) were identified by prolactin experiment. The4miRNAs positively response to prolactin and had potential functions of regulating milk fat metabolism in goat mammary gland.3.200-500bp lengths (including pre-miRNA and flanking sequences) of pri-miR-23a, pri-miR-27a, pri-miR-103and pri-miR-200a were amplified from goat genome, respectively. In comparison to cow, goat pre-miR-27a has one base mutation from A to G on position11of3’. The other goat pre-miRNAs have identical sequences with cow.4recombinant adenovirus plasmids were constructed with their respective pri-miRNA as an inserted sequence. The adenovirus plasmids were transfected to HEK293and packaged in the cells successfully. Adenovirus stably over-expressed miRNAs in GMEC. The miRNA expression mediated by their respective virus in GMEC were3.48-(miR-23a),1.94-(miR-27a),2.92-(miR-103), and2.48-fold (miR-200a) compared with controls (Ad-infected GMECs).4. Indices of milk fat synthesis were measured in Ad-miR-103-infected cells at72h post infection. In comparison to Ad-infected cells, Ad-miR-103-infected cells accumulated more fat droplets. Triglyceride content increased by0.33-fold, total fatty acid content increased by0.16-fold, and unsaturated fatty acid content increased by0.42-fold. Specifically, c9-C18:1and c9,t11-C18:2contents were increased by1.35-and2.16-fold. The expression of FASN, DGAT1, ADRP and SLA27A6associated with milk fat synthesis increased by1.15-,1.21-,18.02-and14.0-fold, respectively. Furthermore, throughout the observation period (0,24,48and72h post infection), expression of PPARy, SREBP-1c, LXRa and the downstream genes of them were all up-regulated. At meanwhile, expression of HSL, ATGL, CPT1and ACOX1 related to fatty acid lipolysis and oxidation was down-regulated. In addition, a strong correlation was identified between miR-103and its host gene PANK3(correlation coefficient R=0.891) in mammary gland, and miR-103can transcriptionally regulate PANK3expression in GMEC.5. Indices of milk fat synthesis were measured in Ad-miR-27a-infected cells at72h post infection. In comparison to Ad-infected cells, fat droplet accumulation in Ad-miR-27a-infected cells was suppressed. The triglyceride content and total fatty acid content both decreased. Specifically, c9,C18:1and c9,t11-C18:2contents decreased by0.64-and0.55-fold, whereas, C16:0and C18:0contents increased by1.19and2.02-fold, resulting the decreased ratio of unsaturated/saturated fatty acid content. Furthermore, throughout the observation period (0,24,48and72h post infection), the expression of PPARy protein was suppressed, and the expression of DGAT1, which is related to triglyceride synthesis and also a downstream gene of PPARy, was down-regulated, too. Additionally, HSL, ATGL and ACOX1expression associated with oxidation and lipolysis was increased, as well as ADRP and TIP47involved in fat droplet formation.6. In comparison to Ad-infected cells, fat droplet and triglyceride contents in Ad-miR-23a-infected cells increased by0.83-and1.21-fold. The expression of FASN, DGAT1and T1P47associated with milk fat synthesis increased by0.82-,0.89-and1.93-fold, whereas, the expression of ATGL related to lipolysis decreased by0.40-fold. Additionally, over-expression of miR-23a up-regulated the expression of ABCA1and ABCG2involved in cholesterol transport. Over-expression of miR-200a in GMEC affected the expression of genes involved in all phases of milk fat synthesis. MiR-200a down-regulated the expression of FASN and ADRP, whereas, up-regulated the expression of SCD, DGAT1and HSL. Furthermore, miR-200a altered the expression of PPARy and SREBP-1c.7. Pearson correlation coefficient (R) was used to analysize the relationship between miRNAs in mammary gland of lactating goats. A strong correlation was identified between3pairs of miRNAs, miR-103and miR-200a (R=0.716), miR-27a and miR-200a (R=0.57), miR-23a and miR-27a (R=0.79). Furthermore, miR-103and miR-200a can mutually down-regulate expression, and the other two pairs can mutually up-regulated expression.