Molecular Characteristics of JAK2 and Its Effect on the Milk Fat and Casein Synthesis of Ovine Mammary Epithelial Cells.

In addition to its association with milk protein synthesis via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, JAK2 also affects milk fat synthesis. However, to date, there have been no reports on the effect of JAK2 on ovine mammary epithelial cells (OMECs), which directly determine milk yield and milk contents. In this study, the coding sequence (CDS) region of ovine JAK2 was cloned and identified and its tissue expression and localization in ovine mammary glands, as well as its effects on the viability, proliferation, and milk fat and casein levels of OMECs, were also investigated. The CDS region of ovine JAK2, 3399 bp in length, was cloned and its authenticity was validated by analyzing its sequence similarity with JAK2 sequences from other animal species using a phylogenetic tree. JAK2 was found to be expressed in six ovine tissues, with the highest expression being in the mammary gland. Over-expressed JAK2 and three groups of JAK2 interference sequences were successfully transfected into OMECs identified by immunofluorescence staining. When compared with the negative control (NC) group, the viability of OMECs was increased by 90.1% in the pcDNA3.1-JAK2 group. The over-expression of JAK2 also increased the number and ratio of EdU-labeled positive OMECs, as well as the expression levels of three cell proliferation marker genes. These findings show that JAK2 promotes the viability and proliferation of OMECs. Meanwhile, the triglyceride content in the over-expressed JAK2 group was 2.9-fold higher than the controls and the expression levels of four milk fat synthesis marker genes were also increased. These results indicate that JAK2 promotes milk fat synthesis. Over-expressed JAK2 significantly up-regulated the expression levels of casein alpha s2 (CSN1S2), casein beta (CSN2), and casein kappa (CSN3) but down-regulated casein alpha s1 (CSN1S1) expression. In contrast, small interfered JAK2 had the opposite effect to JAK2 over-expression on the viability, proliferation, and milk fat and milk protein synthesis of OMECs. In summary, these results demonstrate that JAK2 promotes the viability, proliferation, and milk fat synthesis of OMECs in addition to regulating casein expression in these cells. This study contributes to a better comprehension of the role of JAK2 in the lactation performance of sheep.

MicroRNA-200c Affects Milk Fat Synthesis by Targeting PANK3 in Ovine Mammary Epithelial Cells.

Milk fat is the foremost nutrient of milk and a vital indicator in evaluating milk quality. Accumulating evidence suggests that microRNAs (miRNAs) are involved in the synthesis of milk fat. The miR-200c is closely related to lipid metabolism, but little is known about its effect on the synthesis of milk fat in MECs of ewes. Herein, the effect of miR-200c on the proliferation of ovine mammary epithelial cells (MECs) and its target relationship with a predicted target gene were investigated. The regulatory effects of miR-200c on the expression of the target genes and the content of triglycerides in ovine MECs were further analyzed. The results revealed that the expression level of miR-200c was differentially expressed in both eight tissues selected during lactation and in mammary gland tissues at different physiological periods. Overexpression of miR-200c inhibited the viability and proliferation of ovine MECs, while inhibition of miR-200c increased cell viability and promoted the proliferation of ovine MECs. Target gene prediction results indicated that miR-200c would bind the 3'UTR region of pantothenate kinase 3 (PANK3). Overexpression of miR-200c reduced the luciferase activity of PANK3, while inhibition of miR-200c increased its luciferase activity. These findings illustrated that miR-200c could directly interact with the target site of the PANK3. It was further found that overexpression of miR-200c reduced the expression levels of PANK3 and, thus, accelerated the synthesis of triglycerides. In contrary, the inhibitor of miR-200c promoted the expression of PANK3 that, thus, inhibited the synthesis of triglycerides in ovine MECs. Together, these findings revealed that miR-200c promotes the triglycerides synthesis in ovine MECs via increasing the lipid synthesis related genes expression by targeting PANK3.

MicroRNA-381 Regulates Proliferation and Differentiation of Caprine Skeletal Muscle Satellite Cells by Targeting PTEN and JAG2.

In our previous study, microRNA (miR)-381 was found to be the most down-regulated miRNA in skeletal muscle of Liaoning cashmere goats with higher skeletal muscle mass, but the molecular mechanism involved remains unclear. In this study, primary caprine skeletal muscle satellite cells (SMSCs) were isolated and identified. We investigated the effect of miR-381 on the viability, proliferation and differentiation of caprine SMSCs, and the target relationships of miR-381 with jagged canonical Notch ligand 2 (JAG2) and phosphatase and tensin homolog (PTEN). Cells isolated were positive for SMSC-specific marker protein Pax7. This suggests that purified SMSCs were obtained. The expression level of miR-381 achieved a peak value on day 4 after SMSC differentiation, and miR-381 also significantly increased the expression levels of myogenic differentiation marker genes: myosin heavy chain (MyHC), myogenin (MyoG) and myocyte enhancer factor 2C (MEF2C) in differentiated SMSCs, the area of MyHC-positive myotubes and the myogenic index. These findings suggest that miR-381 promoted myogenic differentiation of caprine SMSCs. The CCK8 assay and EDU staining analysis showed that miR-381 mimic both inhibited the viability of SMSCs and decreased the percentage of EDU-labeled positive SMSCs. In contrast, miR-381 inhibitor had the opposite effect with miR-381 mimic. A dual luciferase reporter assay verified that miR-381 can target JAG2 and PTEN by binding to the 3'-untranslated regions (3'-UTR) of the genes. The transfection of miR-381 mimic into caprine SMSCs resulted in decreases in expression levels of JAG2 and PTEN, while miR-381 inhibitor increased the two target genes in expression. This is the first study to reveal the biological mechanisms by which miR-381 regulates caprine SMSC activities.

Characteristics and Expression of circ_003628 and Its Promoted Effect on Proliferation and Differentiation of Skeletal Muscle Satellite Cells in Goats.

In our previous a study, circ_003628 was one of the most highly expressed circular RNAs (circRNAs) in the Longissimus dorsi muscle of goats found by RNA-seq, suggesting that the circRNA may be important for caprine muscle growth and development. However, there have been no reports describing the molecular mechanisms by which circ_003628 regulates the activities of goat skeletal muscle satellite cells (SMSCs). In this study, reverse transcriptase-PCR (RT-PCR) and DNA sequencing were used to validate the authenticity of circ_003628, and its characteristics, expression profile and effect on goat SMSCs were also studied using real-time quantitative-PCR (RT-qPCR), EdU, CCK-8 and immunofluorescence assays. Circ_003628 is partially originated from 13 exons, 12 introns and 3'-untranslated regions (UTR) of caprine Myosin Heavy Chain 1 (MYH1), and 25 exons and 5' UTR of Myosin Heavy Chain 4 (MYH4), as well as intergenic sequences between the two genes. A total of 77.07% of circ_003628 were located in the nuclei of goat SMSCs, while 22.93% were expressed in the cytoplasm. The circRNAs were only expressed in triceps brachii, quadriceps femoris and longissimus dorsi muscle tissues in nine caprine tissues investigated, with the highest expression level in longissimus dorsi muscle. The expression level of circ_003628 gradually increased during differentiation periods of goat SMSCs and reached the maximum on day 6 after differentiation. The small interfering RNA of circ_003628 (named si-circ_003628) inhibited the viability and proliferation of goat SMSCs, and also decreased the expression of four cell proliferation marker genes: paired box 7 (Pax7), cyclin-dependent kinase 2 (CDK2), CDK4 and CyclinD1 in goat SMSCs. Transfection of si-circ_003628 significantly decreased the area of MyHC-labeled myotubes of goat SMSCs, as well as the expression levels of three differentiation marker genes: myosin heavy chain (MyHC), myogenin (MyoG), and myocyte enhancer factor 2C (MEF2C). These results suggest that circ_003628 promotes the viability, proliferation, and differentiation of goat SMSCs, and they also provide an improved understanding of the roles of circ_003628 in skeletal muscle growth and development in goats.

Tissue-Specific Expression of Circ_015343 and Its Inhibitory Effect on Mammary Epithelial Cells in Sheep.

Circular RNAs (circRNAs) are a kind of non-coding RNA that have an important molecular function in mammary gland development and lactation of mammals. In our previous study, circ_015343 was found to be highly expressed in the ovine mammary gland tissue at the peak-lactation period by using RNA sequencing (RNA-seq). In the present study, the authenticity of circ_015343 was confirmed by using reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and Sanger sequencing. The circ_015343 was derived from the complete 10 exons of aminoadipic semialdehyde synthase (AASS), ranging from exon 2 to exon 11 and mainly located in cytoplasm of ovine mammary epithelial cells. The circRNA was found to be expressed in eight ovine tissues, with the highest expression level in the mammary gland and the least expression in Longissimus dorsi muscle. The circ_015343 had a lower level of expression in a sheep breed with higher milk yield and milk fat content. The disturbed circ_015343 increased the viability and proliferation of the ovine mammary epithelial cells. The inhibition of circ_015343 also increased the expression levels of three milk fat synthesis marker genes: acetyl-coenzyme A carboxylase alpha (ACACA), fatty acid-binding protein 4 (FABP4), and sterol regulatory element-binding protein 1 (SREBP1), as well as three proliferation-related genes: cyclin dependent kinase 2 (CDK2), cyclin dependent kinase 4 (CDK4) and proliferating cell nuclear antigen (PCNA), but decreased the expression level of its parent gene AASS. A circRNA-miRNA-mRNA interaction network showed that circ_015343 would bind some microRNAs (miRNAs) to regulate the expression of functional genes related to the development of mammary gland and lactation. This study contributes to a better understanding of the roles of circ_015343 in the mammary gland of sheep.

Deep Small RNA Sequencing Reveals Important miRNAs Related to Muscle Development and Intramuscular Fat Deposition in Longissimus dorsi Muscle From Different Goat Breeds.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that have been shown to play important post-transcriptional regulatory roles in the growth and development of skeletal muscle tissues. However, limited research into the effect of miRNAs on muscle development in goats has been reported. In this study, Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats with significant phenotype difference in meat production performance were selected and the difference in Longissimus dorsi muscle tissue expression profile of miRNAs between the two goat breeds was then compared using small RNA sequencing. A total of 1,623 miRNAs were identified in Longissimus dorsi muscle tissues of the two goat breeds, including 410 known caprine miRNAs, 928 known species-conserved miRNAs and 285 novel miRNAs. Of these, 1,142 were co-expressed in both breeds, while 230 and 251 miRNAs were only expressed in LC and ZB goats, respectively. Compared with ZB goats, 24 up-regulated miRNAs and 135 miRNAs down-regulated were screened in LC goats. A miRNA-mRNA interaction network showed that the differentially expressed miRNAs would target important functional genes associated with muscle development and intramuscular fat deposition. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the target genes of differentially expressed miRNAs were significantly enriched in Ras, Rap 1, FoxO, and Hippo signaling pathways. This study suggested that these differentially expressed miRNAs may be responsible for the phenotype differences in meat production performance between the two goat breeds, thereby providing an improved understanding of the roles of miRNAs in muscle tissue of goats.

Identification and characterization of circular RNAs in Longissimus dorsi muscle tissue from two goat breeds using RNA-Seq.

Circular RNAs (circRNAs) are a class of non-coding RNA that play crucial roles in the growth and development of skeletal muscle. However, little is known about the role of circRNAs in caprine skeletal muscle. In this study, the size of muscle fiber and the expression profiles of circRNAs were compared in Longissimus dorsi muscle of Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats with significant phenotypic differences in meat production performance, using hematoxylin and eosin staining and RNA-Seq, respectively. The size of muscle fiber in LC goats was larger than those in ZB goats (P < 0.05). A total of 10,875 circRNAs were identified and 214 of these were differentially expressed between the two caprine breeds. The parent genes of differentially expressed circRNAs were mainly enriched in connective tissue development, Rap1, cGMP-PKG, cAMP and Ras signaling pathway. In conclusion, circRNAs may play important roles in skeletal mass, meat production performance and meat quality traits in goats. The results provide an improved understanding of the functions of circRNAs in skeletal muscle development of goats.

Variation in caprine KRTAP1-3 and its association with cashmere fibre diameter.

Keratin-associated proteins (KAPs) are components of cashmere fibres. The gene encoding the KAP1-3 protein (KRTAP1-3) has been described in goats, but little is known about sequence variation in this gene and if it affects cashmere fibre traits. In this study, we used a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to screen for nucleotide sequence variation in caprine KRTAP1-3 in 327 Longdong cashmere goats, then analysed association between the genetic variation that was revealed and some cashmere fibre traits. Six PCR-SSCP patterns representing six different variant sequences of KRTAP1-3 (named A to F) were revealed. Among these variant sequences, seven single nucleotide polymorphisms (SNPs) were detected, with two of them being non-synonymous. Goats with genotype AC had higher mean fibre diameter (MFD) than those with genotype AB (P < 0.001), while goats with genotype AB had higher MFD than those with AA (P < 0.001). The presence of C (P < 0.001) and B (P = 0.006) in a genotype was associated with increased MFD, and together this suggests that variation in caprine KRTAP1-3 affects the key fibre trait of MFD.

MicroRNA-200b Regulates the Proliferation and Differentiation of Ovine Preadipocytes by Targeting p27 and KLF9.

MicroRNAs (miRNAs) are crucial regulatory molecules in lipid deposition and metabolism. However, the effect of miR-200b on the regulation of proliferation and adipogenesis of ovine preadipocytes is unknown in the sheep (Ovis aries). In this study, the expression profiles of miR-200b were investigated in the seven tissues of Tibetan ewes and differentiated preadipocytes. The effect of miR-200b, as well as its target genes p27 and KLF9, on the proliferation of ovine preadipocytes and adipogenesis was also investigated, using cell viability analysis, EdU staining, Oil Red O staining and reverse transcription-quantitative PCR (RT-qRCR). The miR-200b was expressed in all the tissues investigated, and it was highly expressed in lung, liver, subcutaneous adipose and spleen tissues. The expression of miR-200b continuously decreased when the differentiation of ovine preadipocytes initiated. The miR-200b mimic dramatically accelerated the proliferation but inhibited differentiation of ovine preadipocytes. The miR-200b inhibitor resulted in an opposite effect on the proliferation and differentiation of ovine preadipocytes. The dual luciferase reporter assay results showed that miR-200b mimic significantly decreased the luciferase activity of p27 and KLF9 in HEK293 cells transfected with wild-type dual luciferase reporter vectors. This suggests that p27 and KLF9 are the target genes of miR-200b. In over-expressed-p27 preadipocytes, the number of EdU-labeled preadipocytes and the expression levels of proliferation marker genes CDK2, CDK4, CCND1 and PCNA significantly decreased. In addition, the transfection of over-expressed-KLF9 vector into adipocytes remarkably increased the accumulation of lipid droplets and the expression levels of differentiation marker genes aP2, PPARγ, LPL and GLUT4. These results suggest that miR-200b accelerated the proliferation but inhibited the adipogenic differentiation of ovine preadipocytes by targeting p27 and KLF9, respectively.

Variation in a Newly Identified Caprine KRTAP Gene Is Associated with Raw Cashmere Fiber Weight in Longdong Cashmere Goats.

Keratin-associated proteins (KAPs) and keratins determine the physical and chemical properties of cashmere fibers as they are the main components of the fibers. It has been reported that ovine KRTAP1-2 affects clean fleece weight, greasy fleece weight and yield in sheep, but the gene has not been described in goats and its effects on fiber traits are unknown. In this study, we identify the keratin-associated protein 1-2 gene (KRTAP1-2) in the goat genome and describe its effect on cashmere fiber traits in 359 Longdong cashmere goats. Six sequence variants (named CAPHI-KRTAP1-2*A to CAPHI-KRTAP1-2*F) were revealed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. These sequences have the highest homology with ovine KRTAP1-2 sequences. There were a 60-bp deletion, a 15-bp insertion and five single nucleotide polymorphisms (SNPs) including two non-synonymous SNPs in the coding sequence. The caprine KRTAP1-2 gene was expressed in the skin tissue, but a signal was not observed for the kidneys, liver, lungs, spleen, heart and longissimus dorsi muscle. Variation in caprine KRTAP1-2 was found to be associated with raw cashmere fiber weight, but not with fiber diameter and length.

Induction of co-inhibitory molecule CTLA-4 by human papillomavirus E7 protein through downregulation of histone methyltransferase JHDM1B expression.

Human papillomavirus causes various skin diseases and even cancer. Unfortunately, the host immune system often fails to generate effective responses against HPV infection due to the ability of HPV to evade immune-mediated eradication, although the detailed mechanisms by which HPV inhibits host antiviral immunity are not fully understood. In this study, we reported a novel role of HPV E7 oncoprotein in inducing the expression of co-inhibitory molecule cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) in cells of epithelial origin. Mechanistically, HPV E7 protein downregulated the cellular abundance of Jumonji C histone demethylase 1B (JHDM1B), increasing the levels of H3K36 methylation within the promoter region of CTLA-4. Our findings expand the current understanding of HPV-mediated immune evasion mechanisms and may be helpful in developing optimal anti-HPV therapeutic strategies and relevant drugs.

Refractory hypotension in a patient with Wernicke's encephalopathy.

A 57-year-old male patient with gastric carcinoma underwent radical distal gastrectomy type II + Braun anastomosis, and received total parenteral nutrition for 10 days after surgery, followed by small amounts of semi-liquid nutrition for 3 days and liquid nutrition for 2 days. The patient developed refractory hypotension for more than 1 week in the early course of disease, and on Day 15 after surgery presented with characteristic signs of Wernicke's encephalopathy, including diplopia and mental confusion. The hypotension did not improve despite appropriate fluid replacement soon after admission. Treatment with moderate dose of thiamine for 3 months partly relieved ophthalmoplegia and confusion, but not Korsakoff syndrome. This extraordinary presentation with refractory hypotension and the unusual course of the disease encouraged us to present this case.

In vitro study of DNA interaction with melamine and its related compounds.

In vitro studies on the interactions between native herring sperm DNA (HS-DNA) and melamine as well as its related compounds (MARCs), that is, ammeline, ammelide, and cyanuric acid, have been investigated by spectrophotometric, spectrofluorometric, melting temperature, and viscosimetric techniques. It was found that any of the MARCs might interact with HS-DNA by a groove mode of binding via hydrogen bonds. The interaction constants between any of the MARCs and HS-DNA were at 104-105 L mol⁻¹, determined by both spectrophotometric and spectrofluorometric methods. The thermodynamic studies suggested that the interaction processes were exothermic favored (ΔH < 0) and entropy disfavored (ΔS < 0), which indicated that hydrogen bonds might be the main acting force in the binding of MARCs and DNA.