N6-methyladenosine (m6A) is the most common internal modification in mRNAs of all higher eukaryotes. Here we perform two high-throughput sequencing methods, m6A-modified RNA immunoprecipitation sequence (MeRIP-seq) and RNA sequence (RNA-seq) to identify key genes with m6A modification in cashmere fiber growth. A total of 9,085 m6A sites were differentially RNA m6A methylated as reported from by MeRIP-seq, including 7,170 upregulated and 1,915 downregulated. In addition, by comparing m6A-modified genes between the fine-type Liaoning cashmere goat (FT-LCG) and coarse-type Liaoning Cashmere Goat (CT-LCG) skin samples, we obtain 1,170 differentially expressed genes. In order to identify the differently methylated genes related to cashmere fiber growth, 19 genes were selected to validate by performing qRT-PCR in FT-LCG and CT-LCG. In addition, GO enrichment analysis shows that differently methylated genes are mainly involved in keratin filament and intermediate filament. These findings provide a theoretical basis for future research on the function of m6A modification during the growth of cashmere fiber.

Circular RNA (circRNA) is endogenous non-coding RNA (ncRNA) with a covalently closed circular structure. It is mainly generated through RNA alternative splicing or back-splicing. CircRNA is known in the majority of eukaryotes and very stable. However, knowledge of the circRNA involved in regulating cashmere fineness is limited. Skin samples were collected from Liaoning cashmere goats (LCG) and Inner Mongolia cashmere goats (MCG) during the anagen period. For differentially expressed circRNAs, RNA sequencing was performed, and the analysis led to an identification of 17 up-regulated circRNAs and 15 down-regulated circRNAs in LCG compared with MCG skin samples. In order to find the differentially expressed circRNAs in LCG, we carried out qPCRs on 10 candidate circRNAs in coarse type skin of LCG (CT-LCG) and fine type skin of LCG (FT-LCG). Four circRNAs: ciRNA128, circRNA6854, circRNA4154 and circRNA3620 were confirmed to be significantly differential expression in LCG. Also, a regulatory network of circRNAs-miRNAs was bioinformatically deduced and may help to understand molecular mechanisms of potential circRNA involvement in regulating cashmere fineness.

Circular RNAs (CircRNAs) are a type of non-coding RNAs, which contain a covalently closed loop structure without 5′ to 3′ free ends. CircRNAs play essential roles in the regeneration of secondary hair follicle (SHF) and cashmere growth in goats. CircRNA-1926 was previously identified in SHF of cashmere goats, but its potential roles are unclear. In this study, we confirmed the expression of circRNA-1926 in SHF bulge of nine cashmere goats with a significantly higher level at anagen than that of telogen. Through the use of both overexpression and siRNA interference, we showed that circRNA-1926 promoted the differentiation of SHF stem cell into hair follicle lineage in cashmere goats which was evaluated via indictor genes Keratin 7 and Keratin 17. Using RNA pull-down, we found that circRNA-1926 bound with miR-148a/b-3p. Additionally, our data indicated that circRNA-1926 promoted the expression of the CDK19 gene. Using dual-luciferase reporter assays, it was revealed that circRNA-1926 positively regulated the CDK19 expression through miR-148a/b-3p. The results from this study demonstrated that circRNA-1926 contributes the differentiation of SHF stem cells into hair follicle lineages in cashmere goats via sponging miR-148a/b-3p to enhance CDK19 expression.

Circular RNAs (circRNAs), a novel class of non-coding RNAs, can interact with miRNAs through a sequence-driven sponge mechanism, thereby regulating the expression of their downstream target genes. CircRNA-1967 was found in secondary hair follicles (SHFs) of cashmere goats, but its functions are not clear. Here, we showed that both circRNA-1967 and its host gene BNC2 had significantly higher expression in SHF bulge at anagen than those at telogen of cashmere goats. Also, circRNA-1967 participates in the differentiation of SHF stem cells (SHF-SCs) into hair follicle lineage in cashmere goats. RNA pull-down assay verified that circRNA-1967 interacts with miR-93-3p. We also indicated that circRNA-1967 promoted LEF1 expression in SHF-SCs of cashmere goats. By dual-luciferase reporter analysis, we found that circRNA-1967 up-regulated LEF1 expression through the miR-93-3p-mediated pathway. The results from this study demonstrated that circRNA-1967 participated in the differentiation of goat SHF-SCs into hair follicle lineage by sponging miR-93-3p to enhance LEF1 expression. Our founding might constitute a novel pathway for revealing the potential mechanism of the differentiation of SHF-SCs into hair follicle lineage in cashmere goats. Also, these results provided a valuable basis for further enhancing the intrinsic regeneration of cashmere goat SHFs with the formation and growth of cashmere fibers.

Circular RNAs (circRNAs) have stable structures, being a covalently closed loop without 5^′ and 3^′ free ends. They can function as “miRNA sponges” in regulating the expression of their target genes. It was thought that circRNAs are involved in the development of the secondary hair follicle (SHF) in cashmere goats. In our previous investigation, a new circRNA named circRNA-0100 was identified from the SHF of cashmere goats, but its function is unknown. In this work, we found that circRNA-0100 exhibited significantly higher expression at anagen SHF bulge than its counterpart at telogen in cashmere goats. Based on the use of both overexpression and siRNA interference assays, our data indicated that circRNA-0100 promoted the differentiation of cashmere goat SHF stem cells (SHF-SCs) into hair follicle lineage, which was evaluated by analyzing the transcriptional level changes of six indicator genes in SHF-SCs of cashmere goats. Using the RNA pull-down technique, we showed that circRNA-0100 served as “molecular sponges” of miR-153-3p in SHF-SCs. Through the use of dual-luciferase reporter assays, our data indicated that circRNA-0100 positively regulated the transcriptional expression of the KLF5 gene via the miR-153-3p-mediated pathway. Ultimately, we showed that circRNA-0100 promoted the differentiation of SHF-SCs into hair lineage, which might be achieved via sequestering miR-153-3p to heighten the KLF5 expression in SHF-SCs of cashmere goats. Our results provide novel scientific evidence for revealing the potential molecular regulatory mechanisms on the differentiation of SHF-SCs into hair lineage in cashmere goats.

Secondary hair follicles (SHFs) produce the thermoregulatory cashmere of goats. MicroRNAs (miRNAs) play indispensable roles in hair follicle formation and growth. However, most studies examining miRNAs related to cashmere have been performed on goat skin. It remains unclear which miRNAs are highly expressed in SHFs or how miRNAs affect cashmere growth. In the present study, we isolated the SHFs under a dissecting microscope and analyzed the miRNA signatures during annual cashmere growth. Small-RNA sequencing followed by genome-wide expression analysis revealed that early anagen is a crucial phase for miRNA regulation of the cashmere growth, as revealed by two predominant groups of miRNAs. Although they exhibited opposite expression patterns, both groups demonstrated sharp changes of expression when in transit from early anagen to mid-anagen. In addition, we identified 96 miRNA signatures that were differentially expressed between different phases among 376 miRNAs. Functional analysis of the predicted target genes of highly expressed or differentially expressed miRNAs indicated that these miRNAs were involved in signal pathways associated with SHF development, regeneration, and regression. Furthermore, miR-143-3p was preferentially expressed in SHFs and Itga6 was identified as one of targets. The dual-luciferase and in situ hybridization assay demonstrated that miR-143-3p directly repressed the expression of Itga6, suggesting a possible novel role for miR-143-3p in cashmere growth.