Expression Profiling of Preadipocyte MicroRNAs by Deep Sequencing on Chicken Lines Divergently Selected for Abdominal Fatness

<div><p>Through posttranscriptional gene regulation, microRNA (miRNA) is linked to a wide variety of biological processes, including adipogenesis and lipid metabolism. Although miRNAs in mammalian adipogenesis have been worked on extensively, their study in chicken adipogenesis is still very limited. To find miRNAs potentially important for chicken preadipocyte development, we compared the preadipocyte miRNA expression profiles in two broiler lines divergently selected for abdominal fat content, by sequencing two small RNA libraries constructed for primary preadipocytes isolated from abdominal adipose tissues. After bioinformatics analyses, from chicken miRNAs deposited in miRBase 20.0, we identified 225 miRNAs to be expressed in preadipocytes, 185 in the lean line and 200 in the fat line (derived from 208 and 203 miRNA precursors, respectively), which corresponds to 114 miRNA families. The let-7 family miRNAs were the most abundant. Furthermore, we validated the sequencing results of 15 known miRNAs by qRT-PCR, and confirmed that the expression levels of most miRNAs correlated well with those of Solexa sequencing. A total of 33 miRNAs was significantly differentially expressed between the two chicken lines (P<0.05). Gene ontology analysis revealed that they could target genes enriched in the regulation of gene transcription and chromatin function, response to insulin stimulation, and IGF-1 signaling pathways, which could have important roles in preadipocyte development. Therefore, a valuable information and resource of miRNAs on chicken adipogenesis were provided in this study. Future functional investigations on these miRNAs could help explore related genes and molecular networks fundamental to preadipocyte development.</p></div

Expression level of 17 novel miRNAs validated.

<p>Note: * indicates the fold changes of 2^-ΔCt values in the fat line against the lean line.</p><p>Expression level of 17 novel miRNAs validated.</p

qRT-PCR validation of miRNAs in the preadipocytes of the lean and fat chicken lines.

<p>miRNA expression levels were normalized, and were selected at high, intermediate and low levels according to their number of reads by Solexa sequencing. Seven miRNAs (gga-miR-148a, gga-miR-101, gga-miR-100, gga-miR-92, gga-miR-130a, gga-miR-19a and gga-miR-221) with significantly differential expression levels were found (* P<0.05; ** P<0.01). Inset shows the enlarged view of the five lowly expressed miRNAs.</p

Differentially expressed miRNAs.

<p>26 up-regulated miRNAs, and 7 down-regulated miRNAs in the fat chicken line. Fold-change (Y-axis) indicates in log2-scale the fold-changes between the number of reads of miRNAs in the fat chicken lines and the lean chicken line.</p

Enriched biological pathways of genes targeted by 33 up-regulated and down-regulated miRNAs with significantly differential expression level.

<p>Y-axis shows the significance level of enrichment after Bonferroni adjustment.</p

Circular view of miRNAs identified in the chicken genome.

<p>Data tracks viewed from outside inwards: 1) chicken chromosomes; 2) and 3) miRNAs abundantly expressed in the lean and fat chicken lines, respectively; 4) miRNA labels; 5) link lines for miRNA paralogs found to be expressed in chicken preadipocytes. Details on the sequence alignment between miRNA precursors for these miRNA paralogs (chromosome coordinates and sequence identity) can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117843#pone.0117843.s004" target="_blank">S2 Table</a>.</p

The top 10 most abundantly expressed miRNAs.

<p>The top 10 most abundantly expressed miRNAs.</p

Number of reads and expression levels of the 15 miRNAs chosen for qRT-PCR validation.

<p>Number of reads and expression levels of the 15 miRNAs chosen for qRT-PCR validation.</p

Length distribution of small RNA read sequences in fat and lean chicken lines.

<p>Length distribution of small RNA read sequences in fat and lean chicken lines.</p