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Simple Summary: Dermal papilla cells (DPCs) play a key role in the growth and development of hair follicles, and their number can regulate the shape and size of hair. Previous studies found that the BMP7 gene is differentially expressed in Hu sheep lamb skin of different pattern types and plays a key role in the proliferation of Hu sheep DPCs. However, the molecular mechanism of the differential expression of the BMP7 gene remains unclear. Here, we aim to investigate the cause of the BMP7 gene differentially expressed in Hu sheep lamb skin of different pattern types. Our findings demonstrate that DNA methylation modification can affect the expression of the BMP7 gene and can regulate the proliferation and cell cycle of Hu sheep DPCs. Previous studies have shown that the BMP7 gene is differentially expressed in Hu sheep lamb skin of different pattern types, and its expression level is significantly correlated with hair follicle indices of different pattern types, but the molecular mechanism of the differential expression of the BMP7 gene remains unclear. This study investigated the effect of DNA methylation on the transcriptional expression of BMP7. Firstly, we found that the mRNA expression of the BMP7 gene and the activity of the core promoter of the BMP7 gene were upregulated after 5-Aza-Deoxycytidine-induced demethylation treatment using qRT-PCR and double luciferase reporter assay. Then, we found that the proliferation of Hu sheep DPCs in vitro was promoted after 5-Aza-Deoxycytidine-induced demethylation treatment through qRT-PCR, CCK-8, and EdU assay, and that the overexpression of DNMT1 in DPCs induced the opposite effect. In addition, the results of the cell cycle assay reveal that the percentage of cells in the S phase was increased after 5-Aza-Deoxycytidine-induced demethylation treatment, and that the percentage of cells in the S phase was decreased after overexpression of DNMT1 in DPCs. This study indicated that the differential expression of the BMP7 gene in different patterns of Hu sheep lamb skin may be regulated by DNA methylation modification. In addition, DNA methylation can regulate the proliferation and cell cycle of DPCs in Hu sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: Sheep diarrhea is one of the most common illnesses in sheep farming and diarrhea in sheep caused by E. coli occupies a significant proportion of them. E. coli F17 is one of the main members of E. coli causing diarrhea in sheep. This study is devoted to improving the resistance of sheep itself to E. coli F17. This study provides theoretical support for the solution of E. coli F17-caused diarrhea in sheep. Diarrhea is the most common issue in sheep farms, typically due to pathogenic Escherichia coli (E. coli) infections, such as E. coli F17. microRNA, a primary type of non-coding RNA, has been shown to be involved in diarrhea caused by pathogenic E. coli. To elucidate the profound mechanisms of miRNA in E. coli F17 infections, methods such as E. coli F17 adhesion assay, colony counting assay, relative quantification of bacterial E. coli fimbriae gene expression, indirect immune fluorescence (IF), Cell Counting Kit-8 (CCK-8), 5-ethynyl-2′-deoxyuridine (EdU), Western blotting (WB), and scratch assay were conducted to investigate the effect of miR-329b-5p overexpression/knock-down on E. coli F17 susceptibility of sheep intestinal epithelial cells (IECs). The findings indicated that miR-329b-5p enhances the E. coli F17 resistance of sheep IECs to E.coli F17 by promoting adhesion between E. coli F17 and IEC, as well as IEC proliferation and migration. In summary, miR-329b-5p plays a crucial role in the defense of sheep IECs against E. coli F17 infection, providing valuable insights into its mechanism of action. [ABSTRACT FROM AUTHOR]

Simple Summary: Screening for polymorphisms in the promoter region of a functional gene is an effective way to identify useful markers for improving sheep growth and development. Non-SMC condensin I complex subunit G (NCAPG) is a candidate gene linked with sheep growth and development. Its explicit role in muscle development is still unclear, and markers in NCAPG's promoter region have not been explored yet. The goal of this study was to investigate the direct role of NCAPG in regulating myogenic development and the differentiation of myoblasts and explore potential markers in its promoter region in relation to sheep growth and development traits. To achieve this goal, cell proliferation and differentiation after RNA interference with NCAPG were investigated in embryonic myoblasts. In addition, the genetic markers in the promoter region of NCAPG were scanned, and association analysis between the markers and sheep growth and development traits was carried out. The results suggest that interfering with NCAPG inhibits the proliferation and differentiation of myoblasts. Five variants detected in the promoter region of NCAPG were significantly (p < 0.05) associated with sheep growth and development traits. These results provide direct evidence of NCAPG regulating myogenesis and provide useful genetic markers to increase the efficacy of the selection of sheep growth and development traits. Previously, NCAPG was identified as a candidate gene associated with sheep growth traits. This study aimed to investigate the direct role of NCAPG in regulating myogenesis in embryonic myoblast cells and to investigate the association between single-nucleotide polymorphisms (SNPs) in its promoter region and sheep growth traits. The function of NCAPG in myoblast proliferation and differentiation was detected after small interfering RNAs (siRNAs) knocked down the expression of NCAPG. Cell proliferation was detected using CCK-8 assay, EdU proliferation assay, and flow cytometry cell cycle analysis. Cell differentiation was detected via cell immunofluorescence and the quantification of myogenic regulatory factors (MRFs). SNPs in the promoter region were detected using Sanger sequencing and genotyped using the improved multiplex ligation detection reaction (iMLDR®) technique. As a result, a notable decrease (p < 0.01) in the percentage of EdU-positive cells in the siRNA-694-treated group was observed. A significant decrease (p < 0.01) in cell viability after treatment with siRNA-694 for 48 h and 72 h was detected using the CCK-8 method. The quantity of S-phase cells in the siRNA-694 treatment group was significantly decreased (p < 0.01). After interfering with NCAPG in myoblasts during induced differentiation, the relative expression levels of MRFs were markedly (p < 0.05 or p < 0.01) reduced compared with the control group on days 5–7. The myoblast differentiation in the siRNA-694 treatment group was obviously suppressed compared with the control group. SNP1, SNP2, SNP3, and SNP4 were significantly (p < 0.05) associated with all traits except body weight measured at birth and one month of age. SNP5 was significantly (p < 0.05) associated with body weight, body height, and body length in six-month-old sheep. In conclusion, interfering with NCAPG can inhibit the proliferation and differentiation of ovine embryonic myoblasts. SNPs in its promoter region can serve as potential useful markers for selecting sheep growth traits. [ABSTRACT FROM AUTHOR]

Simple Summary: Dermal papilla cells are a vital cell type in the hair follicles. It has been reported that the number of dermal papilla cells could affect the growth and development of hair follicles. Our previous study found that the CRABP2 gene is highly expressed in dermal papilla cells, but its function in dermal papilla cells is still unclear. In this study, we detected the role of the CRABP2 gene in the proliferation of dermal papilla cells and its effect on the Wnt/β-Catenin pathway. Our findings indicate that CRABP2 could promote the proliferation of dermal papilla cells by activating the Wnt/β-Catenin pathway. In our previous study of Hu sheep hair follicles, we found that CRABP2 was highly expressed in DPCs, which suggested that CRABP2 may influence the number of DPCs. In the present study, we aimed to understand the effect of CRABP2 in Hu sheep dermal papilla cells (DPCs). First, we explored the influence of CRABP2 on the ability of Hu sheep DPCs' proliferation. Based on the results obtained from some experiments, such as CCK-8, EDU, qPCR, and Western blot experiment, we found that the overexpression of CRABP2 facilitated the proliferation of DPCs compared to the negative control group. Then, we also detected the effect of CRABP2 on the Wnt/β-catenin pathway based on the important function of the Wnt/β-catenin pathway in hair follicles. The results showed that CRABP2 could activate the Wnt/β-catenin pathway in DPCs, and it rescues the proliferation of DPCs when the Wnt/β-catenin pathway was inhibited. In summary, our findings indicate that CRABP2 is a vital functional gene in the proliferation of Hu sheep DPCs. Our study will be of great use for revealing the roles of CRABP2 in the hair follicles of Hu sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: Escherichia coli (E. coli) F17 is one of the major pathogenic bacteria responsible for diarrhea in farm animals; however, little is known about the biological mechanism underlying E. coli F17 infection. The aim of our study was to reveal the interplay between intestinal genes, metabolites and bacteria in E. coli F17 infected sheep. Our results confirm that the intestinal differ significantly in sheep with different E. coli F17 susceptibility, and integrated omics analyses reveal subsets of potential biomarkers for E. coli F17 infection (i.e., GlcADG 18:0-18:2, ethylmalonic acid and FBLIM1). Our results can help in the development of new insight for the treatment of farm animals infected by E. coli F17. Escherichia coli (E. coli) F17 is one of the most common pathogens causing diarrhea in farm livestock. In the previous study, we accessed the transcriptomic and microbiomic profile of E. coli F17-antagonism (AN) and -sensitive (SE) lambs; however, the biological mechanism underlying E. coli F17 infection has not been fully elucidated. Therefore, the present study first analyzed the metabolite data obtained with UHPLC-MS/MS. A total of 1957 metabolites were profiled in the present study, and 11 differential metabolites were identified between E. coli F17 AN and SE lambs (i.e., FAHFAs and propionylcarnitine). Functional enrichment analyses showed that most of the identified metabolites were related to the lipid metabolism. Then, we presented a machine-learning approach (Random Forest) to integrate the microbiome, metabolome and transcriptome data, which identified subsets of potential biomarkers for E. coli F17 infection (i.e., GlcADG 18:0-18:2, ethylmalonic acid and FBLIM1); furthermore, the PCCs were calculated and the interaction network was constructed to gain insight into the crosstalk between the genes, metabolites and bacteria in E. coli F17 AN/SE lambs. By combing classic statistical approaches and a machine-learning approach, our results revealed subsets of metabolites, genes and bacteria that could be potentially developed as candidate biomarkers for E. coli F17 infection in lambs. [ABSTRACT FROM AUTHOR]

CUT-like homeobox 1 protein (CUX1), also called CUX, CUTL1, and CDP, is a member of the DNA-binding protein homology family. Studies have shown that CUX1 is a transcription factor that plays an important role in the growth and development of hair follicles. The aim of this study was to investigate the effect of CUX1 on the proliferation of Hu sheep dermal papilla cells (DPCs) to reveal the role of CUX1 in hair follicle growth and development. First, the coding sequence (CDS) of CUX1 was amplified by PCR, and then CUX1 was overexpressed and knocked down in DPCs. A Cell Counting Kit-8 (CCK8), 5-ethynyl-2-deoxyuridine (EdU), and cell cycle assays were used to detect the changes in the proliferation and cell cycle of DPCs. Finally, the effects of overexpression and knockdown of CUX1 in DPCs on the expression of WNT10, MMP7, C-JUN, and other key genes in the Wnt/β-catenin signaling pathway were detected by RT-qPCR. The results showed that the 2034-bp CDS of CUX1 was successfully amplified. Overexpression of CUX1 enhanced the proliferative state of DPCs, significantly increased the number of S-phase cells, and decreased the number of G0/G1-phase cells (p < 0.05). CUX1 knockdown had the opposite effects. It was found that the expression of MMP7, CCND1 (both p < 0.05), PPARD, and FOSL1 (both p < 0.01) increased significantly after overexpression of CUX1 in DPCs, while the expression of CTNNB1 (p < 0.05), C-JUN, PPARD, CCND1, and FOSL1 (all p < 0.01) decreased significantly. In conclusion, CUX1 promotes proliferation of DPCs and affects the expression of key genes of the Wnt/β-catenin signaling pathway. The present study provides a theoretical basis to elucidate the mechanism underlying hair follicle development and lambskin curl pattern formation in Hu sheep. [ABSTRACT FROM AUTHOR]

Simple Summary: The Sox18 gene is important for the growth and function of dermal papilla cells, but its effect on the induction ability of dermal papilla cells in Hu sheep is not clear. Through experiments, we analyzed the effect of Sox18 on the induction ability of dermal papilla cells in Hu sheep. The results showed that Sox18 could enhance the induction ability of dermal papilla cells, and regulate the induction ability of dermal papilla cells through the Wnt/β-catenin signal pathway. We believe that Sox18 is an important gene that affects the induction ability of Hu sheep's dermal papilla cells, and these findings may provide new avenues for studying the growth and development mechanism of hair. Sox18 is a developmental gene that encodes transcription factors. It has been indicated as be a key gene affecting the growth and development of hair follicles, in which dermal papilla cells (DPCs) have been demonstrated to play an important role through their ability to induce the formation of hair follicles. Pre-laboratory studies have found that Sox18 is differentially expressed in the dermal papilla cells of different pattern types of Hu sheep. We speculated that Sox18 plays an important role in the dermal papilla cells of Hu sheep. In our study, we analyzed the effect of Sox18 on the induction ability of DPCs in order to elucidate the function and molecular mechanism of Sox18 in the DPCs of Hu sheep. We first identified the expression of Sox18 in the DPCs of Hu sheep by immunofluorescence staining. We then used alkaline phosphatase staining, cell morphology observations and RT-PCR to detect the effect of Sox18 on the induction of DPCs after overexpression of or interference with Sox18. We also used RT-PCR, WB and immunofluorescence staining to detect the effect of Sox18 on the Wnt/β-catenin signal pathway in DPCs. We found that Sox18 was specifically expressed in the DPCs of Hu sheep, and that Sox18 could enhance the alkaline phosphatase activity in the DPCs of Hu sheep and accelerate cell agglutination. The results of RT-PCR revealed that Sox18 promoted the mRNA expression of Versican, HHIP and FGFRI, and inhibited the mRNA expression of BMP4 and WIF1. Further studies showed that Sox18 promoted the expression of β-catenin and activated the Wnt/β-catenin signal pathway in DPCs. When the Wnt/β-catenin signal pathway of DPCs was activated, the induction ability of DPCs was enhanced. Overall, we believe that Sox18 could enhance the induction ability of DPCs in Hu sheep and regulate the induction ability of DPCs through the Wnt/β-catenin signal pathway. [ABSTRACT FROM AUTHOR]

Previous genome-wide association studies (GWAS) have found that LAP3 may have the potential function to impact sheep muscle development. In order to further explore whether LAP3 expression has an important role in the development of sheep embryonic myoblasts, we conducted the spatiotemporal expression profile analysis of LAP3 at the tissue and cellular level. Then we used small interfering RNA and eukaryotic recombinant vectors to perform gain/loss-of-function analysis of LAP3. CCK-8 detection, EdU staining, and flow cytometry were used to investigate the impact of LAP3 knockdown or overexpression on the proliferation of embryonic myoblasts. In addition, cell phenotype observation, MyHC indirect immunofluorescence, and quantitative detection of the expression changes of myogenic regulatory factors (MRFs) were used to explore the effect of LAP3 on myogenic differentiation. The results showed that the LAP3 expression level in muscle tissue of fetuses was significantly higher than that in newborn lambs and adult sheep, and its expression level on day 3 of differentiation was also significantly higher than that in the proliferation phase and other differentiation time points. LAP3 silencing could significantly increase cell viability and EdU-positive cells, as well as prolonging the length of S phase of myoblasts to promote proliferation, while the results were reversed when LAP3 was overexpressed. Moreover, LAP3 silencing significantly hindered myotube formation and down-regulated the expression levels of MRFs from day 5 to day 7 of terminal differentiation, while the results were reversed when LAP3 was highly expressed. Overall, our results suggested that the expression of LAP3 impacts on the development of sheep embryonic myoblasts which provides an important theoretical basis for molecular breeding of meat production in sheep. [ABSTRACT FROM AUTHOR]

Early growth response factor 1 (EGR1) is a zinc-finger transcription factor that plays a vital role in the development of hair follicles. According to our previous studies, EGR1 is a transcriptional promoter of the bone morphogenetic protein 7 (BMP7), a candidate gene involved in the proliferation of dermal papilla cells. Since hair follicles are the basis of lambskin pattern formation and dermal papilla cells (DPCs) act on hair follicle growth, in order to elucidate the role of EGR1 and hair follicles, this study aimed to investigate the biological role of EGR1 in DPCs. In our study, the EGR1 coding sequence (CDS) region was firstly cloned by polymerase chain reaction, and bioinformatics analysis was performed. Then, the function of EGR1 was detected by 5-ethynyl-2'-deoxyuridine (EDU) and Cell Counting Kit-8 (CCK8), and Western blot (WB) was conducted to analyze the cellular effect of EGR1 on DPCs. The proliferative effect of EGR1 on DPCs was also further confirmed by detecting its expression by qPCR and WB on marker genes of proliferation, including PCNA and CDK2. The sequence of the EGR1 CDS region of a lamb was successfully cloned, and its nucleic acid sequence was analyzed and found to be highly homologous to Rattus norvegicus, Mus musculus, Bos taurus and Homo sapiens. Predictive analysis of the protein encoded by EGR1 revealed that it is an extra-membrane protein, and not a secretory protein, with subcellular localization in the nucleus and cytoplasm. The proliferative effect of DPCs was significantly stronger (p < 0.01) in EGR1 up-regulated DPCs compared to the controls, while the opposite result was observed in EGR1 down-regulated DPCs. Markers of proliferation including PCNA and CDK2 also appeared to be differentially upregulated in EGR1 gene overexpression compared to the controls, with the opposite result in EGR1 gene downregulation. In summary, our study revealed that EGR1 promotes the proliferation of DPCs, and we speculate that EGR1 may be closely associated with hair follicle growth and development. [ABSTRACT FROM AUTHOR]

Simple Summary: Diarrhea and vomiting caused by Escherichia coli (E. coli) F17 are considered significant threats to animal farming. In the present study, RNA-Seq was performed to investigate the potential circRNA and miRNA biomarkers for E. coli F17-antagonism (AN) and -sensitive (SE) lambs. The results indicated that circRNA and miRNA expression is closely associated with the susceptibility of E. coli F17 in lambs. Numbers of circRNAs and miRNAs may serve as potential biomarkers for intestinal inflammatory response against E. coli F17 infection. Our study can provide a preliminary understanding of the underlying mechanisms of intestinal immunity. It has long been recognized that enterotoxigenic Escherichia coli (ETEC) is the major pathogen responsible for vomiting and diarrhea. E. coli F17, a main subtype of ETEC, is characterized by high morbidity and mortality in young livestock. However, the transcriptomic basis underlying E. coli F17 infection has not been fully understood. In this study, RNA sequencing was performed to explore the expression profiles of circRNAs and miRNAs in the jejunum of E. coli F17-antagonism (AN) and -sensitive (SE) lambs. A total of 16,534 circRNAs and 271 miRNAs (125 novel miRNAs and 146 annotated miRNAs) were screened, and 214 differentially expressed (DE) circRNAs and 53 DE miRNAs were detected between the AN and SE lambs (i.e., novel_circ_0025840, novel_circ_0022779, novel_miR_107, miR-10b). Functional enrichment analyses showed that source genes of DE circRNAs were mainly involved in metabolic-related pathways, while target genes of DE miRNAs were mainly enriched in the immune response pathways. Then, a two-step machine learning approach combining Random Forest (RF) and XGBoost (candidates were first selected by RF and further assessed by XGBoost) was performed, which identified 44 circRNAs and 39 miRNAs as potential biomarkers (i.e., novel_circ_0000180, novel_circ_0000365, novel_miR_192, oar-miR-496-3p) for E. coli infection. Furthermore, circRNA-related and lncRNA-related ceRNA networks were constructed, containing 46 circRNA-miRNA-mRNA competing triplets and 630 lncRNA-miRNA-mRNA competing triplets, respectively. By conducting a serious of bioinformatic analyses, our results revealed important circRNAs and miRNAs that could be potentially developed as candidate biomarkers for intestinal inflammatory response against E. coli F17 infection; our study can provide novel insights into the underlying mechanisms of intestinal immunity. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) is of great importance to muscle growth and development, including the regulation of the proliferation and differentiation of skeletal muscle satellite cells (SMSCs). In our research group's previous study, we found that miR-181a is differentially expressed in the longissimus dorsi muscle of Hu sheep at different stages. We speculated that miR-181a may participate in the growth and development process of Hu sheep. To understand the mechanism of miR-181a regulating the growth and development of Hu sheep skeletal muscle, we extracted skeletal muscle satellite cells from the longissimus dorsi muscle of 3-month-old Hu sheep fetuses and performed a series of experiments. Our results showed that miR-181a suppressed SMSCs' proliferation using QRT-PCR, Western blot, CCK-8, EDU, and Flow cytometry cycle tests. In addition, QRT-PCR, Western blot, and immunofluorescence indicated that miR-181a facilitated the differentiation of SMSCs. Then, we used dual-luciferase reporter gene detection, QRT-PCR, and Western blot to find that the Yes1-related transcription regulator (YAP1) is the target gene of miR-181a. Our study supplies a research basis for understanding the regulation mechanism of miR-181a on the growth of Hu sheep skeletal muscle. [ABSTRACT FROM AUTHOR]

Hair follicle development is closely associated with wool curvature. Current studies reveal the crucial role of microRNAs (miRNAs) in hair follicle growth and development. However, few studies are known regarding their role in wool curvature. To reveal the potential roles of miRNAs in Hu sheep lambskin with different patterns, a total of 37 differentially expressed (DE) miRNAs were identified in hair follicles between small waves (SM) and straight wool (ST) groups using RNA-seq. Through functional enrichment and miRNA-mRNA co-expression analysis, some key miRNAs (oar-miR-143, oar-miR-200b, oar-miR-10a, oar-miR-181a, oar-miR-10b, oar-miR-125b, etc.) and miRNA-mRNA pairs (miR-125b target CD34, miR-181a target FGF12, LMO3, miR-200b target ZNF536, etc.) were identified. Though direct or indirect ways affecting hair follicle development, these miRNAs and mRNAs may have possible effects on wool curvature, and this study thus provides valuable insight on potential pattern formation. [ABSTRACT FROM AUTHOR]

Bone morphogenetic proteins (BMPs) are the structurally similar and highly conserved type of functional proteins that play an important role in hair follicle growth and development. BMP7 was a differentially expressed gene in different patterns of Hu sheep lambskin identified using Agilent microarray. Since hair follicle is the basis of pattern formation of lambskin, and its growth and development is governed by dermal papilla cells (DPCs), to clarify the role of BMP7 and hair follicle, our study was designed to investigate the regulation between BMP7 and DPCs. Firstly, the CDS region of BMP7 was cloned by 3'Race and PCR in Hu sheep and performed serious of bioinformatic analysis. Then, the effects of BMP7 on DPCs were analyzed after overexpression and interference of BMP7 in dermal papilla cells by CCK8, EdU, and PI assay. Additionally, qPCR was also conducted to clarify the relationship between BMP7 and the TGF-β/Smad signaling pathway. A total of 1296 bp of the BMP7 CDS region sequence was sucessfully cloned in Hu sheep, encoding a signal peptide of 431 amino acids, molecular weight was 49,316.9 Da and the isoelectric point (Pi) was 7.75. Nucleotide sequencing analysis of BMP7 revealed that Hu sheep had high homology with Bos taurus, Homo sapiens, and Canis lupus familiaris. Structure domain prediction showed that TGF-β superfamily domain exist between 330th–431th amino acid, BMP7 protein is a secreted protein. In BMP7 up-regulated DPCs, DPCs proliferation rate and cell cycle were significantly higher than that of NC group (p < 0.05). Meanwhile, the expression level of Smad3, Smad4, Samd6, and TGF-β1 in TGF-β/Smad signaling pathway were significantly lower than that in NC group (p < 0.05). In BMP7 down-regulated DPCs, it presented the opposite result. In conclusion, our study showed that BMP7 had a positive effect on DPCs by accelerating the proliferation and cell cycle of DPCs, and hypothesized that regulate hair follicles growth and development via TGF-β/Smad signaling pathway. These findings may provide a synergistic target for the subsequent research of hair follicle growth and development. [ABSTRACT FROM AUTHOR]

Simple Summary: The meat production of Hu sheep affects the mutton supply, and how to increase the meat production is the primary focus of genetic breeding. Therefore, in order to explore the key factors affecting the proliferation and differentiation of Hu sheep skeletal muscle cells, we performed functional verification of miRNA at the cellular level. Our findings are helpful to clarify the molecular regulation mechanism of proliferation and differentiation of skeletal muscle cells, which will benefit the molecular breeding of Hu sheep. The growth and development of skeletal muscle require a series of regulatory factors. MiRNA is a non-coding RNA with a length of about 22 nt, which can inhibit the expression of mRNA and plays an important role in the growth and development of muscle cells. The role of miR-22-3p in C2C12 cells and porcine skeletal muscle has been reported, but it has not been verified in Hu sheep skeletal muscle. Through qPCR, CCK-8, EdU and cell cycle studies, we found that overexpression of miR-22-3p inhibited proliferation of skeletal muscle cells (p < 0.01). The results of qPCR and immunofluorescence showed that overexpression of miR-22-3p promoted differentiation of skeletal muscle cells (p < 0.01), while the results of inhibiting the expression of miR-22-3p were the opposite. These results suggested that miR-22-3p functions in growth and development of sheep skeletal muscle cells. Bioinformatic analysis with mirDIP, miRTargets, and RNAhybrid software suggested IGFBP3 was the target of miR-22-3p, which was confirmed by dual-luciferase reporter system assay. IGFBP3 is highly expressed in sheep skeletal muscle cells. Overexpression of IGFBP3 was found to promote proliferation of skeletal muscle cells indicated by qPCR, CCK-8, EdU, and cell cycle studies (p < 0.01). The results of qPCR and immunofluorescence experiments proved that overexpression of IGFBP3 inhibited differentiation of skeletal muscle cells (p < 0.01), while the results of interfering IGFBP3 with siRNA were the opposite. These results indicate that miR-22-3p is involved in proliferation and differentiation of skeletal muscle cells by targeting IGFBP3. [ABSTRACT FROM AUTHOR]

Simple Summary: This study was conducted to explore the antibacterial ability of sheep β-defensin 2 (SBD-2) against E. coli F17 infection in ovine intestinal epithelial cells (OIECs). Our data revealed that E. coli F17 induces SBD-2 expression in OIECs in vitro, which appears to be mediated through the activation of the signaling pathways NF-κB and MAPK. Our results provide a novel insight for the functionality of SBD-2, which could be useful for developing anti-infective drugs and/or breeding for E. coli diarrhea disease-resistant sheep. Escherichia coli (E. coli) F17 is a member of enterotoxigenic Escherichia coli, which can cause massive diarrhea and high mortality in newborn lambs. β-defensin is mainly produced by the epithelial tissue of the gastrointestinal tract in response to microbial infection. However, the molecular mechanism of sheep β-defensin 2 (SBD-2) against E. coli F17 remains unclear. This study aims to reveal the antibacterial ability of SBD-2 against E. coli F17 infection in sheep. Firstly, we established the culture system of ovine intestinal epithelial cells (OIECs) in vitro, treated with different concentrations of E. coli F17 for an indicated time. Secondly, we performed RNA interference and overexpression to investigate the effect of SBD-2 expression on E. coli F17 adhesion to OIECs. Finally, inhibitors of NF-κB and MAPK pathways were pre-treated to explore the possible relationship involving in E. coli F17 infection regulating SBD-2 expression. The results showed that E. coli F17 markedly (p < 0.01) upregulated the expression levels of SBD-2 mRNA and protein in a concentration- and time-dependent manner. Overexpression of SBD-2 contributed to enhancing E. coli F17 resistance in OIECs, while silencing SBD-2 dramatically improved the adhesion of E. coli F17 to OIECs (p < 0.05 or p < 0.01). Furthermore, E. coli F17 stimulated SBD-2 expression was obviously decreased by pre-treatment with NF-κB inhibitor PDTC, p38 MAPK inhibitor SB202190 and ERK1/2 MAPK inhibitor PD98095 (p < 0.05 or p < 0.01). Interestingly, adhesion of E. coli F17 to OIECs were highly enhanced by pre-treated with PDTC, SB202190 and PD98095. Our data suggested that SBD-2 could inhibit E. coli F17 infection in OIECs, possibly through NF-κB and MAPK signaling pathways. Our results provide useful theoretical basis on developing anti-infective drug and breeding for E. coli diarrhea disease-resistant sheep. [ABSTRACT FROM AUTHOR]

Wool curvature is the determining factor for lambskin quality of Hu lambs. However, the molecular mechanism of wool curvature formation is not yet known. miRNA has been proved to play an important role in hair follicle development, and we have discovered a differentially expressed miRNA, miR-143, in hair follicles of different curl levels. In this study, we first examined the effects of miR-143 on the proliferation and cell cycle of dermal papilla cells using CCK8, EdU and flow cytometry and showed that miR-143 inhibited the proliferation of dermal papilla cells and slowed down the cell cycle. Bioinformatics analysis was performed to predict the target genes KRT71 and CUX1 of miR-143, and both two genes were expressed at significantly higher levels in small waves than in straight lambskin wool (p < 0.05) as detected by qPCR and Western blot (WB). Then, the target relationships between miR-143 and KRT71 and CUX1 were verified through the dual-luciferase assay in 293T cells. Finally, after overexpression and suppression of miR-143 in dermal papilla cells, the expression trend of CUX1 was contrary to that of miR-143. Meanwhile, KRT71 was not detected because KRT71 was not expressed in dermal papilla cells. Therefore, we speculated that miR-143 can target CUX1 to inhibit the proliferation of dermal papilla cells, while miR-143 can target KRT71 to regulate the growth and development of hair follicles, so as to affect the development of hair follicles and ultimately affect the formation of wool curvature. [ABSTRACT FROM AUTHOR]

Homozygosity of long sequence genotypes are a result of parents transmitting identical haplotypes, which can be used to estimate their auto-zygosity. Therefore, we used high-density SNP Chip data to characterize the auto-zygosity of each breed according to the occurrence and distribution of runs of homozygosity (ROH). Subsequently, we identified the genomic regions with high runs of homozygosity frequencies within individuals of each breed. We selected 96 sheep samples from five local Chinese sheep breeds belonging to different geographical locations. We identified 3046 ROHs within the study breed individuals, among which the longer segments (>1–5 Mb) were dominant. On average, ROH segments covered about 12% of the genomes; the coverage rate of OAR20 was the lowest and that of OAR2 was the highest. The distribution analysis of runs of homozygosity showed that the detected ROH mainly distributed between >26 and 28 Mb. The Hetian and Hu sheep showed the lowest ROH distribution. The estimation of homozygosity level reflects the history of modern and ancient inbreeding, which may affect the genomes of Chinese indigenous sheep breeds and indicate that some animals have experienced recent self-pollination events (Yabuyi, Karakul and Wadi). In these sheep breeds, the genomic regions were assumed to be under selection signatures frequently in line with long ROH. These regions included candidate genes associated with disease resistance traits (5S_rRNA), the innate and adaptive immune response (HERC2 and CYFIP1), digestion and metabolism (CENPJ), growth (SPP1), body size and developments (GJB2 and GJA3). This study highlighted new insights into the ROH patterns and provides a basis for future breeding and conservation strategies of Chinese sheep breeds. [ABSTRACT FROM AUTHOR]

Through long term natural and artificial selection, domestic sheep (Ovis aries) have become adapted to a diverse range of agro-ecological environments and display multiple phenotypic traits. Characterization of diversity and selection signature is essential for genetic improvement, understanding of environmental adaptation, as well as utilization and conservation of sheep genetic resources. Here, we aimed to assess genomic diversity, population structure, and genomic selection among five Chinese native sheep breeds using 600K high density SNP genotypes. A total of 96 animals of the five breeds were selected from different geographical locations with extremely dry or humid conditions. We found a high proportion of informative SNPs, ranging from 93.3% in Yabuyi to 95.5% in Wadi, Hu, and Hetian sheep. The average pairwise population differentiation (FST) between the breeds was 0.048%, ranging from 0.022% to 0.054%, indicating their low to moderate differentiation. PCA, ADMIXTURE, and phylogenetic tree analyses revealed a clustering pattern of the five Chinese sheep breeds according to their geographical distribution, tail type, coat color, body size, and breeding history. The genomic regions under putative selection identified by FST and XP-EHH approaches frequently overlapped across the breeds, and spanned genes associated with adaptation to extremely dry or humid environments, innate and adaptive immune responses, and growth, wool, milk, and reproduction traits. The present study offers novel insight into genomic adaptation to dry and humid climates in sheep among other domestic animals and provides a valuable resource for further investigation. Moreover, it contributes useful information to sustainable utilization and conservation of sheep genetic resources. [ABSTRACT FROM AUTHOR]

Simple Summary: Bone morphogenetic protein 7 (BMP7) is one of the largest secretory signal conductive molecules and is in the TGF-β superfamily. It plays an important role in the growth and development of hair follicles. We cloned the proximal promoter of the BMP7 gene for bioinformatics analysis. Dual-luciferase reporter system and overexpression were used to analyze the key regions and transcription factor binding sites. There was high activity between −758 bp and −545 bp in the core region of the gene and a possible binding site for transcription factors SP1 and EGR1. The BMP7 gene is involved in the growth and development of hair follicles but its regulation mechanism is unclear. We studied the regulation mechanism of the BMP7 promoter by cloning the proximal promoter of BMP7 for bioinformatics analysis. A series of missing vectors was then constructed for dual-fluorescein activity detection based on the bioinformatics analysis results. We tested transcription-factor binding-site mutations and transcription factor over-expression to analyze the transcriptional regulation principle of the BMP7 promoter region. The upstream transcriptional regulatory region of the BMP7 gene proximal promoter was predicted by bioinformatics. There were −1216 bp to −1166 bp and −632 bp to −582 bp transcription initiation sites in the upstream transcriptional regulatory region of the BMP7 gene proximal promoter. The CpG islands' distribution showed that there were many CpG islands at −549 bp to 1 bp. A dual-luciferase assay revealed high activity between −758 bp and −545 bp in the core region and a possible binding site for transcription factors SP1 and EGR1. The transcriptional activity of BMP7 was significantly decreased in the transcriptional regulatory region of the BMP7 after EGR1 and SP1 mutation. Transcription was significantly enhanced by over expression of the EGR1 transcription factor, which strongly suggests that EGR1 and SP1 play important roles in BMP7 regulation. [ABSTRACT FROM AUTHOR]

Sox18 is a developmental gene that encodes transcription factors. It has been indicated as be a key gene affecting the growth and development of hair follicles, in which dermal papilla cells (DPCs) have been demonstrated to play an important role through their ability to induce the formation of hair follicles. Pre-laboratory studies have found that Sox18 is differentially expressed in the dermal papilla cells of different pattern types of Hu sheep. We speculated that Sox18 plays an important role in the dermal papilla cells of Hu sheep. In our study, we analyzed the effect of Sox18 on the induction ability of DPCs in order to elucidate the function and molecular mechanism of Sox18 in the DPCs of Hu sheep. We first identified the expression of Sox18 in the DPCs of Hu sheep by immunofluorescence staining. We then used alkaline phosphatase staining, cell morphology observations and RT-PCR to detect the effect of Sox18 on the induction of DPCs after overexpression of or interference with Sox18 . We also used RT-PCR, WB and immunofluorescence staining to detect the effect of Sox18 on the Wnt/β-catenin signal pathway in DPCs. We found that Sox18 was specifically expressed in the DPCs of Hu sheep, and that Sox18 could enhance the alkaline phosphatase activity in the DPCs of Hu sheep and accelerate cell agglutination. The results of RT-PCR revealed that Sox18 promoted the mRNA expression of Versican , HHIP and FGFRI , and inhibited the mRNA expression of BMP4 and WIF1 . Further studies showed that Sox18 promoted the expression of β-catenin and activated the Wnt/β-catenin signal pathway in DPCs. When the Wnt/β-catenin signal pathway of DPCs was activated, the induction ability of DPCs was enhanced. Overall, we believe that Sox18 could enhance the induction ability of DPCs in Hu sheep and regulate the induction ability of DPCs through the Wnt/β-catenin signal pathway.