Long noncoding RNAs (lncRNAs) are a novel class of eukaryotic transcripts. They are thought to act as a critical regulator of protein-coding gene expression. Herein, we identified and characterized 13 putative lncRNAs from the expressed sequence tags from secondary hair follicle of Cashmere goat. Furthermore, we investigated their transcriptional pattern in secondary hair follicle of Liaoning Cashmere goat during telogen and anagen phases. Also, we generated intracellular regulatory networks of upregulated lncRNAs at anagen in Wnt signaling pathway based on bioinformatics analysis. The relative expression of six putative lncRNAs (lncRNA-599618, -599556, -599554, -599547, -599531, and -599509) at the anagen phase is significantly higher than that at telogen. Compared with anagen, the relative expression of four putative lncRNAs (lncRNA-599528, -599518, -599511, and -599497) was found to be significantly upregulated at telogen phase. The network generated showed that a rich and complex regulatory relationship of the putative lncRNAs and related miRNAs with their target genes in Wnt signaling pathway. Our results from the present study provided a foundation for further elucidating the functional and regulatory mechanisms of these putative lncRNAs in the development of secondary hair follicle and cashmere fiber growth of Cashmere goat.

MicroRNAs (miRNAs) are endogenous small noncoding RNA molecules that negatively regulate gene expression. Herein, we investigated a selective number of miRNAs for their expression in skin tissue of Liaoning Cashmere goat during hair follicle cycles, and their intracellular regulatory networks were constructed based on bioinformatics analysis. The relative expression of six miRNAs (mir-103-3p, -15b-5p, 17-5p, -200b, -25-3p, and -30c-5p) at anagen phase is significantly higher than that at catagen and/or telogen phases. In comparison to anagen, the relative expression of seven miRNAs (mir-148a-3p, -199a-3p, -199a-5p, -24-3p, -30a-5p, -30e-5p, and -29a-3p) was revealed to be significantly up-regulated at catagen and/or telogen stages. The network analyses of miRNAs indicated those miRNAs investigated might be directly or indirectly involved in several signaling pathways through their target genes. These results provided a foundation for further insight into the roles of these miRNAs in skin tissue of Liaoning Cashmere goat during hair follicle cycles.

Bone morphogenetic protein 4 (BMP4) is a member of the bone morphogenetic protein family (BMPs). It is involved in the development and cycle of hair follicle, as well as, is thought to be a potential candidate gene for cashmere traits in goats. In the present study, we isolated and characterized a full-length open reading frame (ORF) of BMP4 cDNA from the skin tissue of Liaoning cashmere goat, and investigated the transcriptional pattern and methylation status of BMP4 gene in skin tissue of this breed during different stages of hair follicle cycle. The sequence analysis indicated that the isolated cDNA was 1264-bp in length containing a complete ORF of 1230-bp. It encoded a precursor peptide of 409 amino acids with a signal peptide of 19 amino acids. The structural analysis indicated that goat BMP4 contains typical TGF-β propeptide and TGF-β domains. In skin tissue, BMP4 is generally transcribed in an ascendant pattern from anagen to telogen. The methylation level of 5' flanking regulatory region of BMP4 gene might be involved in its mRNA expression in skin tissue: a higher BMP4 methylation level in skin coincides with a lower expression of BMP4 mRNA. These results from the present work provided a foundation for further insight into the functional and regulatory characteristics of BMP4 in the development and cycle of hair follicle in Liaoning Cashmere goat.

Homeobox protein Hox-C8 (HOXC8) is a member of Hox family. It is expressed in the dermal papilla of the skin and is thought to be associated with the hair inductive capacity of dermal papilla cells. In the present study, we isolated and characterized a full-length open reading frame of HOXC8 cDNA from the skin tissue of Liaoning cashmere goat, as well as, established a phylogenetic relationship of goat HOXC8 with that of other species. Also, we investigated the effect of methylation status of HOXC8 exon 1 at anagen secondary hair follicle on the cashmere fiber traits in Liaoning cashmere goat. The sequence analysis indicated that the obtained cDNA was 1134-bp in length containing a complete ORF of 729-bp. It encoded a peptide of 242 amino acid residues in length. The structural analysis indicated that goat HOXC8 contained a typical homeobox domain. The phylogenetic analysis revealed that Capra hircus HOXC8 had a closer genetic relationship with that of Ovis aries, followed by Bos Taurus and Bubalus bubalis. The methylation analysis suggested that the methylation degree of HOXC8 exon 1 in anagen secondary hair follicle might be involved in regulating the growth of cashmere fiber in Liaoning cashmere goat. Our results provide new evidence for understanding the molecular structural and evolutionary characteristics of HOXC8 in Liaoning cashmere goat, as well as, for further insight into the role of methylation degree of HOXC8 exon 1 regulates the growth of cashmere fiber in goat.

ABSTRACT:The microRNAs are non-coding RNA molecules of approximately 20-22 nucleotides that are found to be implicated in a wide range of physiological processes. In this study, the suitability of 10 candidate reference RNAs was evaluated for microRNA expression data in the skin tissue of Liaoning cashmere goat including 1 small nuclear RNA (snRNA; RNU6B), 1 small nucleolar RNA (snoRNA; Z30), 1 rRNA (5S), 1 transfer RNA (tRNA; MettRNA), and 6 microRNAs (miR; let-7d-5p, miR-15a-5p, miR-26a-5p, miR-125a-5p, miR-214-3p, and miR-221-3p). Based on geNorm and NormFinder algorithms, we identified let-7d-5p, miR-26a-5p, and miR-15a-5p as the most stable reference RNAs. Also, three reference RNAs (let-7d-5p, miR-26a-5p, and miR-15a-5p) were sufficient for the normalization of microRNA expression data in the skin of this breed. We further assessed the suitability of let-7d-5p, miR-26a-5p, and miR-15a-5p in a combination as reference RNAs through detecting the relative expression of miR- 24-3p, miR-29a-3p, miR-145a-5p, and miR-205-5p as putative genes of interest. Significant differences were revealed in the relative expression of miR- 24-3p, miR-29a-3p, miR-145a-5p, and miR-205-5p at telogen stage of hair follicle cycle when a combination of let-7d-5p, miR-26a-5p, and miR-15a-5p vs a single let-7d-5p were used as reference RNA. Based on the results from this study, we suggested that the combination of let-7d-5p, miR-26a-5p, and miR-15a-5p as normalizers for microRNA expression data would be more reliable than that of single let-7d-5p, and the geometric mean of these three microRNAs (let-7d-5p, miR-26a-5p, and miR15a-5p) can be used for the normalization of microRNAs expression data in the skin of Liaoning cashmere goat.