Analysis of miRNAs and their target genes associated with lipid metabolism in duck liver

Abstract

Fat character is an important index in duck culture that linked to local flavor, feed cost and fat intake for costumers. Since the regulation networks in duck lipid metabolism had not been reported very clearly, we aimed to explore the potential miRNA-mRNA pairs and their regulatory roles in duck lipid metabolism. Here, Cherry-Valley ducks were selected and treated with/without 5% oil added in feed for 2 weeks, and then fat content determination was performed on. The data showed that the fat contents and the fatty acid ratios of C17:1 and C18:2 were up-regulated in livers of oil-added ducks, while the C12:0 ratio was down-regulated. Then 21 differential miRNAs, including 10 novel miRNAs, were obtain from the livers by sequencing, and 73 target genes involved in lipid metabolic processes of these miRNAs were found, which constituted 316 miRNA-mRNA pairs. Two miRNA-mRNA pairs including one novel miRNA and one known miRNA, N-miR-16020-FASN and gga-miR-144-ELOVL6, were selected to validate the miRNA-mRNA negative relation. And the results showed that N-mir-16020 and gga-miR-144 could respectively bind the 3'-UTRs of FASN and ELOVL6 to control their expressions. This study provides new sights and useful information for future research on regulation network in duck lipid metabolism.

Figure 1

Read width frequency distributions (a) and volcano plot (b) of miRNAs from CG and EG livers. CG and EG represent control group and experimental group, respectively. This also applies to the following Figures and Tables.

Figure 2. Heatmap of differentially expressed miRNAs in CG and EG livers.

The prefix “N-” means predicted novel miRNAs.

Figure 3. Regulation networks of the differentially expressed miRNAs and their predicted target genes in duck lipid metabolism.

Figure 4. Target gene Numbers of the differential miRNAs in duck lipid metabolic processes.

Figure 5

Relative expression levels of N-miR-16020 and gga-miR-144 (a) and their chosen target genes FASN and ELOVL6 (b) in CG and EG. Expression levels of the two miRNAs and their chosen target genes were determined by qRT-PCR in the CG and EG liver samples, normalized to those of U6 and β-actin respectively, and then shown as mean ± SD. Each transfection was performed in triplicates. *P < 0.05, **P < 0.01.

Figure 6

Dual-Luciferase reporter system validations of gga-miR-144-ELOVL6 (a) and N-miR-16020-FASN (b). Recombined Dual –Luciferase reporter vectors with 3′-UTR of target genes were cotransfected with miRNAs, while cotransfection with negative mimics and transfection without mimics were settled as negative control (NC) and blank control (BC), respectively. Each transfection was performed in 6 replicates. The firefly: Renilla luciferase activities were calculated and shown as mean ± SD. *P < 0.05, **P < 0.01. The information of sequence analysis between miRNA seed regions and target 3′-UTRs was placed at the bottom.