ABSTRACT: Hu sheep lambskin is a unique white lambskin from China that exhibits three types of flower patterns, including small waves, medium waves, and large waves, with small waves considered the best quality. However, our understanding of the molecular mechanism underlying flower pattern formation in Hu sheep lambskin is limited. The aim of the present study was to further explore the relevance between candidate microRNAs (miRNAs) and developmental characteristics of hair follicles and screen miRNAs for later functional validation. Herein, we employed Illumina Hiseq 2500 to identify differentially expressed miRNAs in hair follicles of different flower patterns with small, medium, and large waves to construct a comprehensive sequence database on the mechanism of hair follicle development. Paraffin sections of lambskin tissue were prepared to assess the structure of different hair follicles. Expression levels of candidate miRNAs in different flower patterns were analyzed by relative quantitation using real-time PCR, combined with histological observation and micro-observation technologies, and the correlation between expression levels of candidate miRNAs and histological properties of hair follicles was analyzed by using SPSS 17.0. A total of 522 differentially expressed miRNAs were identified, and RNA-seq analysis detected 7,266 target genes in different groups of flower patterns. Gene ontological analysis indicated these target genes were mainly involved in cell proliferation, differentiation, growth, apoptosis, and ion transport, and 14 miRNAs, including miR-143, miR-10a, and let-7 were screened as candidate miRNAs in Hu sheep hair follicle growth and development. In the same field of vision, variance analysis showed that the number of secondary follicles in small waves was significantly larger than that in large and medium waves (P<0.01); the diameter of the primary and secondary follicles in large waves was respectively larger than those in medium and small waves (P<0.01). Combined with correlation analysis between miRNA expression and histological properties of hair follicles, highly significant differences in miRNA-143 expression levels between large and small waves were observed (P<0.01), and significant differences in the miRNA-10a expression levels between large and small waves (P<0.05) and in let-7i expression levels between large and medium waves were observed (P<0.05). Significant differences in the expression of novel miRNAs of NW_004080184.1_6326 between medium and large waves were detected (P<0.05), and highly significant differences between medium and small waves were observed (P<0.01). Highly significant differences in the expression level of NW_004080165.1_8572 between medium and large and small waves (P<0.01), in that of NW_004080181.1_3961 between medium and small waves (P<0.01), and in that of NW_004080190.1_13733 between medium and large waves were observed, whereas no significant differences in the other miRNAs among large, medium, and small waves were detected. Overall, the present study showed that miRNA-143, miRNA-10a, let-7i, NW_004080184.1_6326, NW_004080165.1_8572, NW_004080181.1_3961, and NW_004080190.1_13733 could be considered as important candidate genes, indicating these seven miRNAs may play significant roles in hair follicle growth and development in Hu sheep lambskin.
