Abstract
MicroRNAs (miRNAs) play critical roles in the control of skin and hair follicle development, epidermal homeostasis and pigmentation. However, the roles of miRNAs in the skins of rabbits with different hair types are unclear. In this study, we profiled miRNAs in the skins of long and short haired rabbits by Illumina deep sequencing. The dataset was compared with known mammalian miRNAs in miRBase 21.0. In total, 118 miRNAs were found to be differentially expressed between the two different rabbit types, of which 94 were upregulated, and 24 were downregulated in the skin of short haired vs. long haired rabbits. The expression levels of five randomly selected miRNAs detected by quantitative real-time PCR indicated that the expression patterns were consistent with Illumina sequencing results. What’s more, bioinformatics analysis showed that miR-125a might target Wnt2, an important modulator for hair follicle development. To test whether Wnt2 is a target of miR-125a, luciferase reporter vector (pMir-report-Wnt2-3′-UTR-WT) and its substitution mutant (pMir-report-Wnt2-3′-UTR-MUT) were constructed. Co-transfection and reporter enzyme assays showed that compared with control (miR-125a NC transfection), miR-125a mimics transfection significantly inhibited the reporter luciferase activities expressed by pMir-report-Wnt2-3′-UTR-WT, while transfection of miR-125a inhibitors increased reporter enzyme activities. RT-PCR and Simple Western analysis found that Wnt2 mRNA and protein levels were induced or repressed by miR-125a overexpression or inhibition, respectively. Moreover, the mRNA expression levels of genes in Wnt signaling pathway, such as CTNNB1, LEF-1, PPARD and TGFB1, were also significantly changed (P < 0.05), consistent with Wnt2. It indicated that the regulation of Wnt2 expression by miRNAs may depend on the transcriptional degradation. The results will help to further understand the role of miRNAs in hair follicle development and the genetic mechanism underlying hair length phenotype.
Highlights
MicroRNAs are an emerging class of regulators that control post-transcriptional processes and could be potential drug targets as well as potential sites of phenotypic regulation (Lewis et al, 2003; Alvarez-Garcia and Miska, 2005)
To systematically identify small RNAs and changes in the expression levels of miRNAs in the long and short haired rabbits, we purified and sequenced small RNAs isolated from rabbit skin
The lengths of most of the small RNAs were between 18 and 24 nt, with the most common size of the small RNAs being 22 nt, which accounted for 30% ∼ 40% of the small RNAs in the skins of long and short haired rabbits (Figure 1)
Summary
MicroRNAs (miRNAs) are an emerging class of regulators that control post-transcriptional processes and could be potential drug targets as well as potential sites of phenotypic regulation (Lewis et al, 2003; Alvarez-Garcia and Miska, 2005). Some miRNAs have been found to play critical roles in cell differentiation and the proliferation of skin and hair follicles and their target genes play. Rabbit miRNAs have been identified by small RNA sequence reads with SOLiD and Illumina platforms (Li et al, 2011), currently, only 12 entries representing hairpin precursor miRNAs and 21 entries for mature miRNA products in rabbits have been identified and deposited in the public miRNA database miRBase This number is far less than for Mus musculus (1,193 precursors, 1,920 mature), Rattus norvegicus (495 precursors, 807 mature) and Homo sapiens (1,881 precursors, 2,603 mature). Given the identification of the roles of miRNAs in hair follicle development, identifying differentially expressed miRNAs is a key step in investigating the function of miRNAs in rabbit skin
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