N6-methyladenosine (m6A) is the most common internal modification in mRNAs of all higher eukaryotes. Here we perform two high-throughput sequencing methods, m6Amodified RNA immunoprecipitation sequence (MeRIP-seq) and RNA sequence (RNAseq) 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 m6Amodified 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.

Objective:The objective of this study was to investigate the effects of N 6 -Methyladenosine modification-circRNA-zinc finger protein 638 (m 6 A-circRNA-ZNF638) on the induced activation of secondary hair follicle (SHF) stem cells with its potential mechanisms in cashmere goats. Methods: The m 6 A modification of ZNF638 was analyzed using Methylation Immunoprecipitation (Me-RIP) with real-time quantitative PCR (RT-qPCR) technique in SHF stem cells. The effects of circRNA-ZNF638 on the induced activation of SHF stem cells in m 6 A dependence were evaluated through the overexpression of circRNA-ZNF638/its m 6 A-deficient mutants in circRNA-ZNF638 knockdown SHF stem cells. The competitive binding of miR-361-5p to circRNA-ZNF638/Wnt5a 3′untranslated region (3′-UTR) was analyzed through Dual-luciferase reporter assay. Results:The m 6 A-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, m 6 A-circRNA-ZNF638 sponged miR-361-5p to heighten the transcriptional expression of Wnt5a gene in SHF-stem cells. We further demonstrated that the

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.

Competitive endogenous RNA (ceRNA) is a transcript that can be mutually regulated at the post-transcriptional level by competing shared miRNAs. The ceRNA network connects the function of protein-encoded mRNA with the function of non-coding RNA, such as microRNA (miRNA), long non-coding RNA (lncRNA), and circular RNA (circRNA). However, compared with the ceRNA, the identification and combined analysis of lncRNAs, mRNAs, miRNAs, and circRNAs in the cashmere fineness have not been completed. Using RNA-seq technology, we first identified the miRNAs presented in Liaoning Cashmere Goat (LCG) skin, and then analyzed the mRNAs, lncRNAs, circRNAs expressed in LCG and Inner Mongolia cashmere goat (MCG) skin. As a result, 464 known and 45 new miRNAs were identified in LCG skin. In LCG and MCG skin, 1222 differentially expressed mRNAs were identified, 170 differentially expressed lncRNAs and 32 differentially expressed circRNAs were obtained. Then, qRT-PCR was used to confirm further the representative lncRNAs, mRNAs, circRNAs and miRNAs. In addition, miRanda predicted the relationships of ceRNA regulatory network among lncRNAs, circRNAs, miRNAs and mRNAs, the potential regulatory effects were investigated by Go and KEGG analysis. Through the screening and analysis of the results, the ceRNA network regulating cashmere fineness was constructed. LncRNA MSTRG14109.1 and circRNA452 were competed with miRNA-2330 to regulated the expression of TCHH, KRT35 and JUNB, which may provide a potential basis for further research on the process of regulating the cashmere fineness.

Cashmere fineness is one of the important factors determining cashmere quality; however, our understanding of the regulation of cashmere fineness at the cellular level is limited. Here, we used single-cell RNA sequencing and computational models to identify 13 skin cell types in Liaoning cashmere goats. We also analyzed the molecular changes in the development process by cell trajectory analysis and revealed the maturation process in the gene expression profile in Liaoning cashmere goats. Weighted gene co-expression network analysis explored hub genes in cell clusters related to cashmere formation. Secondary hair follicle dermal papilla cells (SDPCs) play an important role in the growth and density of cashmere. ACTA2, a marker gene of SDPCs, was selected for immunofluorescence (IF) and Western blot (WB) verification. Our results indicate that ACTA2 is mainly expressed in SDPCs, and WB results show different expression levels. COL1A1 is a highly expressed gene in SDPCs, which was verified by IF and WB. We then selected CXCL8 of SDPCs to verify and prove the differential expression in the coarse and fine types of Liaoning cashmere goats. Therefore, the CXCL8 gene may regulate cashmere fineness. These genes may be involved in regulating the fineness of cashmere in goat SDPCs; our research provides new insights into the mechanism of cashmere growth and fineness regulation by cells.

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