Abstract: 【Objective】 In the present study, high-throughput sequencing and bioinformatics analysis were initially employed to identify differentially expressed microRNAs (miRNAs) in hair follicles of different flower patterns with small, medium, and large waves of Hu sheep. Then 14 candidate miRNAs were screened. The purpose of this study was to study the amount of 14 nominated miRNA expression in different pattern groups and to find the correlation of different hair follicle index, and to further explore the relevance between candidate miRNAs and developmental characteristics of hair follicles and screen miRNAs for later functional validation.【Method】Firstly, utilizing high-throughput sequencing filtrated differentially expressed miRNAs in hair follicles of different flower patterns with small, medium, and large waves of Hu sheep. To confirm the reliability of sequencing results, five miRNAs were selected for RT-PCR verification. Paraffin sections of lambskin tissue were prepared to assess the structure of different hair follicles. Expression levels of 14 candidate miRNAs in different groups were analyzed by relative quantitation using fluorescent quantitative PCR. Combined with histological observation and microscopic observation technique, the correlation between expression levels of 14 candidate miRNAs and histological properties of hair follicles was analyzed by using SPSS 17.0.【Result】The distribution of hair follicles was mainly in the form of hair follicle groups, which distributed as a group of 3. The diameter of the primary follicles was larger than that of secondary follicles regardless of large, medium, or small waves. In the same field of view, no significant differences in primary follicles among the large, medium and small wave lambskins were observed (P＞0.05); the number of secondary follicles in the small waves was significantly higher than that in large and medium waves (P＜0.01), whereas the number of secondary follicles in the medium wave was similar to that in the small waves (P＞0.05). Consequently, the total number of hair follicles in the small waves was higher than that in the large and medium waves in the same unit area. The diameter of the primary and secondary follicles in the large waves were significantly higher than that in medium and small waves (P＜0.01) and the diameter of primary and secondary hair follicles were not significantly different between the medium and small waves groups (P＞0.05). The level of expression of miRNA-143 in the large waves was significantly lower than that in the large waves (P＜0.01). The expression level of miRNA-10a in the small waves was significantly higher than that in the large and medium waves (P＜0.05) and that of let-7i was significantly higher in the medium waves compared with that in the large waves (P＜0.05). In the differentially expressed novel miRNAs, the expression level of NW_004080184.1_6326 in medium waves was significantly lower than that in the large and small waves, respectively (P＜0.05; P＜0.01). The expression of NW_004080165.1_8572 in the medium waves was significantly different compared with that in the large and small waves (P＜0.01). NW_004080181.1_3961 was expressed at a higher level in small waves than in medium waves (P＜0.01). Highly significant differences in NW_004080190.1_13733 expression was observed between medium and large waves (P＜0.01). No significant differences in the expression levels of the rest of the miRNAs were observed among the large, medium, and small waves. The relative expression levels of 14 miRNAs were correlated with some indicators of hair follicles, indicating that all 14 miRNAs might be involved in the growth and development of hair follicles.【Conclusion】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 might play significant roles in hair follicle growth and development in Hu sheep lambskin, which would be confirmed in subsequent experiments.

Key words: Hu sheep lambskin, hair follicles, miRNA, floral pattern, hair follicle development