Knockdown of miR-361-5p promotes the induced activation of SHF-stem cells through FOXM1 mediated Wnt/ß-catenin pathway in cashmere goats.

The inducing activation event of secondary hair follicle (SHF)-stem cells is considered a key biological process in the SHF regeneration, and the morphogenesis of cashmere fiber in cashmere goats. The miR-361-5p was essentially implicated in the induced activation of SHF-stem cells of cashmere goats, but its functional mechanisms are unclear. Here, we confirmed miR-361-5p was significantly downregulated in anagen SHF bugle of cashmere goats compared with that at telogen, and miR-361-5p expression was significantly lower in SHF-stem cells after activation than its counterpart before activation. Further, we found that miR-361-5p could negatively regulate the induced activation event of SHF-stem cells in cashmere goats. Mechanistically, through dual-luciferase reporter assays, miR-361-5p specifically bound with FOXM1 mRNA in SHF-stem cells of cashmere goats and negatively regulated the expression of FOXM1 gene. Also, through overexpression/knockdown analysis of FOXM1 gene, our results indicated that FOXM1 upregulated the expression of Wnt/ß-catenin pathway related genes in SHF-stem cells. Moreover, based on TOP/FOP-flash Wnt report assays, the knockdown of miR-361-5p promotes the Wnt/ß-catenin pathway activation through upregulating the FOXM1 expression in SHF-stem cells. Finally, we demonstrated that miR-361-5p negatively regulated the induced activation of SHF-stem cells through FOXM1 mediated Wnt/ß-catenin pathway in cashmere goats.

Association analysis for SNPs of KRT26 and TCHH genes with cashmere production performance, body measurement traits and milk production traits in Liaoning cashmere goats.

Cashmere fineness is getting thicker, which is one of the key problems in cashmere breeding, however, there have been no systematic studies on the molecular regulation of cashmere fineness. The aim of this study was to investigate the relationship between KRT26 and TCHH gene polymorphism and production performance in Liaoning cashmere goats (LCG). The potential single nucleotide polymorphisms (SNPs) of LCG were detected by sequence alignment and PCR-Seq polymorphism of KRT26 and TCHH genes and analyzed the effect of SNPs on production performance by SPSS software. Two SNPs sites (A559T and A6839G) of two genes were detected. The AA genotype of KRT26 A559T locus was the dominant genotype. AG and GG at TCHH A6839G locus were the dominant genotypes. AAAA was the dominant haplotype combination. The results showed that KRT26 and TCHH genes were associated with cashmere fineness of LCG, and A559T (AA) and A6839G (GG) genotypes were the preferred marker genotypes for cashmere fineness, which provided more theoretical basis for further research on cashmere fineness.

Analysis of m6A methylation in skin tissues of different sex Liaoning cashmere goats.

N6-methyladenosine (m6A) is the most frequent internal modification of mRNA and lncRNA in eukaryotes. We used two high-throughput sequencing method, m6A-seq and RNA-seq to identify pivotal m6A-modified genes in cashmere fineness and fiber growth. 8062 m6A peaks were detected by m6A-seq, including 2157 upregulated and 6445 downregulated. Furthermore, by comparing m6A-modified genes of the male Liaoning Cashmere Goat (M-LCG) and female Liaoning Cashmere Goat (F-LCG) skin tissues, we get 862 differentially expressed m6A-modified genes. To identify differently expressed m6A genes associated with cashmere fineness, 11 genes were selected for validation using real time fluorescent quantitative PCR in M-LCG and F-LCG. This study provides an acadamic basis on the molecular regulation mechanism of m6A modification in cashmere growth process.

Association analysis for SNPs of MSTN and IGFBP-3 genes with body size and other production traits in Liaoning Cashmere Goats.

Liaoning cashmere goat (LCG) have tall bones, high cashmere production and outstanding meat production performance. In recent years, good breeding progress has not been made in terms of body size, meat yield, milk yield and other properties in terms of production. The study focused on the correlation between the SNPs of MSTN and IGFBP-3 genes with the body size performance, cashmere production and milk performance. The MSTN and IGFBP-3 gene sequence alignment and PCR-Seq polymorphism were used to detect the potential SNPs, and the correlation with production performance was analyzed by SPSS and SHEsis software. The results showed that the TT genotype at the T1662G locus of the MSTN gene is dominant and has significant advantages in body measurements such as sacrum height, chest width, and waist height. The C allele at the C4021T locus of IGFBP-3 gene shows an advantage in the body measurement performance. Among the haplotype combinations, H2H2:TGTC is preponderant combination for body size performance, H2H2:TGTC and H1H2:TGCC are preponderant combinations for cashmere production performance, H1H3:GGCC is preponderant combination for milk production performance. It may be a molecular marker for future selection and breeding.

High-throughput analysis of lncRNA in cows with naturally infected Staphylococcus aureus mammary gland.

LncRNA (long non-coding RNA) is an RNA molecule with a length between 200 and 100,000 nt. It does not encode proteins and is involved in a variety of intracellular processes, becoming a research hotspot of genetics. To identify key lncRNAs associated with dairy mastitis, we collected mammary epithelial tissue samples of Normal disease-free Holstein cows (HCN) and unhealthy Holstein cows with Staphylococcus aureus (HCU) and performed RNA sequencing (RNA-seq) on the samples. A total of 270 differentially expressed lncRNAs and 500 differentially expressed mRNAs were identified by high-throughput sequencing and bioinformatics analysis. Furthermore, Hydrolase activity is the most enriched in GO, and ErbB signaling pathway is significantly enriched in KEGG. In addition, through qPCR validation of 5 candidate lncRNAs in HCN and HCU, four differentially expressed lncRNAs MSTRG.498, MSTRG57.1, MSTRG.41.1 and MSTRG 124.1 were confirmed to have significant differentially expressed in cow mastitis. Also, lncRNA MSTRG.498 and its target gene, SMC4, might directly or indirectly play a role in cow mastitis. The regulatory network of lncRNA-miRNA-mRNA has been inferred from a bioinformatics perspective, which may assist understand the underlying molecular mechanism of lncRNAs involved in regulating mastitis in cows. Our findings will provide meaningful resources for further research on the regulatory function of lncRNAs in cow mastitis.

N6-Methyladenosine modification (m6A) of circRNA-ZNF638 contributes to the induced activation of SHF stem cells through miR-361-5p/Wnt5a axis in cashmere 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.

CeRNA regulates network and expression and SNP effect on NFKBIA of cashmere fineness.

In this study, a total of 1140 Liaoning Cashmere Goats (LCG) were genotyped for single nucleotide polymorphism (SNP) of NFKBIA gene. There are 15 SNPs and 7 genotypes have been found, and G1547A (GG) genotype has been associated with cashmere fineness and cashmere yield. An integrated ceRNA regulatory network of NFKBIA gene was made. To prove NFKBIA and these non-coding RNAs (ncRNAs) may be related to cashmere fineness, we performed qPCR on these ncRNA in LCG coarse type skin (CT-LCG) and LCG fine type skin (FT-LCG). The result of qPCR showed lncRNA XLOC_011060 and ciRNA452 are at high expression level in CT-LCG, all miRNAs appear high expressed in FT-LCG, and mir-93 was the most significant difference between CT-LCG and FT-LCG. In addition, five miRNAs were selected for qPCR in different genotypes. The qPCR results showed that mir-93 might negatively regulate cashmere fineness and mir-17-5p may play a positive role in regulating cashmere fineness of individuals with G1355A (AG) genotype. These results demonstrated that NFKBIA gene is associated with cashmere fineness of LCG and G1547A (GG) genotype is the preferred marker genotype for cashmere fineness.

High-throughput analysis of CircRNA in cows with naturally infected Staphylococcus aureus mammary gland.

Circular RNAs (CircRNA) are a special type of non-coding RNA molecule with a closed ring structure and are not affected by RNA exonucases. It has stable expression, is not easy to degrade, and exists in most eukaryotes. However, circRNA regulation of cow mastitis has not been widely recognized. Mammary epithelial tissues were collected from healthy Holstein cows (HCN) and mastitis Holstein cows (HCU). RNA sequencing (RNA SEQ) was performed for the differentially expressed circRNAs, and analysis results showed that 19 differentially expressed circRNAs were identified in HCN and HCU, among which 6 circRNAs were up-regulated and 13 circRNAs were down-regulated. We randomly selected nine circRNAs for Q-PCR verification, and the results showed consistent expression. Three circRNAs: circRNA2860, circRNA5323 and circRNA4027 were confirmed to be significantly differentially expressed circRNAs in cow mastitis. Also, their host genes TRPS1, SLC12A2 and MYH11 might be directly or indirectly play a role in cow mastitis. Furthermore, RNA polymerase transcription factor binding and tight junction are most enriched in GO and KEGG pathways, respectively. In addition, the regulatory network of circRNA-miRNA has been inferred from a bioinformatics perspective, which may help to understand the underlying molecular mechanism of circRNAs involved in regulating mastitis in cows.

Identification and Molecular Analysis of m6A-circRNAs from Cashmere Goat Reveal Their Integrated Regulatory Network and Putative Functions in Secondary Hair Follicle during Anagen Stage.

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.

Single-Cell Sequencing Reveals Differential Cell Types in Skin Tissues of Liaoning Cashmere Goats and Key Genes Related Potentially to the Fineness of Cashmere Fiber.

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.

Screening of cashmere fineness-related genes and their ceRNA network construction in cashmere 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.

Comprehensive analysis of circRNAs from cashmere goat skin by next generation RNA sequencing (RNA-seq).

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.

m6A Methylation Analysis of Differentially Expressed Genes in Skin Tissues of Coarse and Fine Type Liaoning Cashmere Goats.

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.