The Inner Mongolia cashmere goat is an excellent local breed in China. According to the characteristics of wool quilts, the Inner Mongolia cashmere goat can be divided into three types: a long-hair type (hair length of >22 cm), a short-hair type (hair length of ≤13 cm), and an intermediate type (hair length of >13 cm and ≤22 cm). It is found that hair length has a certain reference value for the indirect selection of other important economic traits of cashmere. In order to explore the molecular mechanisms and related regulatory genes of the different hair types, a weighted gene coexpression network analysis (WGCNA) was carried out on the gene expression data and phenotypic data of 12-month-old Inner Mongolia cashmere goats with a long-hair type (LHG) and a short-hair type (SHG) to explore the coexpression modules related to different coat types and nine candidate genes, and detect the relative expression of key candidate genes. The results showed that the WGCNA divided these genes into 19 coexpression modules and found that there was a strong correlation between one module and different hair types. The expression trends of this module’s genes were different in the two hair types, with high expression in the LHG and low expression in the SHG. GO functions are mainly concentrated in cellular components, including intermediate filaments (GO:0005882), intermediate filament cytoskeletons (GO:0045111), and cytoskeletal parts (GO:0044430). The KEGG pathway is mainly enriched in arginine as well as proline metabolism (chx00330) and the MAPK signaling pathway (chx04010). The candidate genes of the different hair types, including the KRT39, KRT74, LOC100861184, LOC102177231, LOC102178767, LOC102179881, LOC106503203, LOC108638293, and LOC108638298 genes, were screened. Through qRT-PCR, it was found that there were significant differences in these candidate genes between the two hair types, and most of them had a significant positive correlation with hair length. It was preliminarily inferred that these candidate genes could regulate the different hair types of cashmere goats and provide molecular markers for hair growth.

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.

N⁶-methyladenosine (m⁶A) is the most abundant modification in linear RNA molecules. Over the last few years, interestingly, many circRNA molecules are also found to have extensive m⁶A modification sites with temporal and spatial specific expression patterns. To date, however, little information is available concerning the expression profiling and functional regulatory characteristics of m⁶A modified circRNAs (m⁶A-circRNAs) in secondary hair follicles (SHFs) of cashmere goats. In this study, a total of fifteen m⁶A-circRNAs were identified and characterized in the skin tissue of cashmere goats. Of these, six m⁶A-circRNAs were revealed to have significantly higher expression in skin at anagen compared with those at telogen. The constructed ceRNA network indicated a complicated regulatory relationship of the six anagen up-regulated m⁶A-circRNAs through miRNA mediated pathways. Several signaling pathways implicated in the physiological processes of hair follicles were enriched based on the potential regulatory genes of the six anagen up-regulated m⁶A-circRNAs, such as TGF-beta, axon guidance, ribosome, and stem cell pluripotency regulatory pathways, suggesting the analyzed m⁶A-circRNAs might be essentially involved in SHF development and cashmere growth in cashmere goats. Further, we showed that four m⁶A-circRNAs had highly similar expression trends to their host genes in SHFs of cashmere goats including m⁶A-circRNA-ZNF638, -TULP4, -DNAJB6, and -CAT. However, the expression patterns of two m⁶A-circRNAs (m⁶A-circRNA-STAM2 and -CAAP1) were inconsistent with the linear RNAs from their host genes in the SHFs of cashmere goats. These results provide novel information for eluci-dating the biological function and regulatory characteristics of the m⁶A-circRNAs in SHF development and cashmere growth in goats.

Objective: The objective of this study was to investigate the effects of N6-Methyladenosine modification-circRNA-zinc finger protein 638 (m6A-circRNA-ZNF638) on the induced activation of secondary hair follicle (SHF) stem cells with its potential mechanisms in cashmere goats.Methods: The m6A modification of ZNF638 was analyzed using methylation immunoprecipitation with real-time quantitative polymerase chain reaction technique in SHF stem cells. The effects of circRNA-ZNF638 on the induced activation of SHF stem cells in m6A dependence were evaluated through the overexpression of circRNA-ZNF638/its m6Adeficient mutants in circRNA-ZNF638 knockdown SHF stem cells. The competitive binding of miR-361-5p to circRNA-ZNF638/Wnt5a 3′- untranslated region was analyzed through Dual-luciferase reporter assay.Results: The m6A-circRNA-ZNF638 had significantly higher transcription at anagen SHF bulge of cashmere goats compared with that at telogen, as well as it positively regulated the induced activation of SHF-stem cells in cashmere goats. Mechanismly, m6A-circRNA-ZNF638 sponged miR-361-5p to heighten the transcriptional expression of Wnt5a gene in SHFstem cells. We further demonstrated that the internal m6A modification within circRNAZNF638 is required for mediating the miR-361-5p/Wnt5a pathway to regulate the induced activation of SHF stem cells through an introducing of m6A-deficient mutant of circRNAZNF638.Conclusion: The circRNA-ZNF638 contributes the proper induced activation of SHF-stem cells in cashmere goats in m6A-dependent manner through miR-361-5p/Wnt5a axis.

Objective: The objective of this study was to identify the N6-methyladenosine (m6A)- circHECA molecule in secondary hair follicles (SHFs) of cashmere goats, and generate its potential regulatory network, as well as explore the potential relationship between transcriptional pattern of m6A-circHECA and promoter methylation of its host gene (HECA).Methods: The validation of circHECA m6A sites was performed using methylation immunoprecipitation (Me-RIP) along with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technique. The nucleus and cytoplasm localizations of m6AcircHECA were performed using SHF stem cells of cashmere goats with RT-qPCR analysis. Based on in-silico analysis, the regulatory networks of m6A-circHECA were generated with related signal pathway enrichment. The methylation level of promoter region of m6A-circHECA host gene (HECA) was assessed by the bisulfite sequencing PCR (BSPPCR) technique.Results: The m6A-circHECA was confirmed to contain four m6A modification sites including m6A-213, m6A-297, m6A-780, and m6A-927, and it was detected mainly in cytoplasm of the SHF stem cells of cashmere goats. The integrated regulatory network analysis showed directly or indirectly complex regulatory relationships between m6A-circHECA of cashmere goats and its potential target molecules: miRNAs, mRNAs, and proteins. The regulatory network and pathway enrichment indicated that m6A-circHECA might play multiple roles in the SHF physiology process of cashmere goats through directly or indirectly interacting or regulating its potential target molecules. A higher methylation level of promoter region of HECA gene in SHFs of cashmere goats might cause the lower expression of m6A-circHECA.Conclusion: The m6A-circHECA might play multiple roles in SHF physiology process of cashmere goats through miRNA mediated pathways along with directly or indirectly interaction with its target proteins. The promoter methylation of m6A-circHECA host gene (HECA) most likely was implicated in its expression inhibition in SHFs of cashmere goats.

The cashmere, a kind of nature protein fiber, is one of the main use of cashmere goats. The induced activation of secondary hair follicle (SHF) stem cells by the dermal papilla cell-derived signals is a key biological process for the morphogenesis and growth of cashmere fiber in cashmere goats. Previously, the circRNA-ERCC6 (circERCC6) was identified from cashmere goat SHFs; however, its biological significance is unclear in the SHF physiology process of cashmere goats. In this study, we found that circERCC6 exhibited significantly higher expression at anagen SHF bulge compared with the counterpart of telogen and harbored three m⁶A modified sites (named m⁶A-685, m⁶A-862, and m⁶A-995) through methylation immunoprecipitation using a real-time quantitative polymerase chain reaction (Me-RIP-qPCR) technique. The knockdown experiments of circERCC6 in SHF stem cells showed that circERCC6 positively regulates the induced activation of SHF stem cells in cashmere goats. Through a dual-luciferase reporter assay, we demonstrated that m⁶A-modified circERCC6 (m⁶A-circERCC6) sponged miR-412-3p to upregulate the expression of BNC2 mRNA in SHFstem cells. Through m⁶A-deficient mutant assay in circERCC6 knockdown SHF stem cells, we further showed that m⁶A modification within circERCC6 is required to mediate the miR-412-3p/BNC2 axis to finally promote the proper induced activation of SHF stem cells in cashmere goats.