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.

Identification and Characterization of Circular RNAs (circRNAs) Using RNA-Seq in Two Breeds of Cashmere Goats.

Circular RNA (circRNA) is a type of non-coding RNA generated from back-splicing the reactions of linear RNA. It plays an important role in various cellular and biological processes. However, there are few studies about the regulatory effect of circRNAs on cashmere fiber traits in cashmere goats. In this study, the expression profiles of circRNAs in skin tissue were compared between Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats, with a significant difference in cashmere fiber yield, cashmere fiber diameter, and cashmere fiber color, using RNA-seq. A total of 11,613 circRNAs were expressed in the caprine skin tissue, and their type, chromosomal distribution, and length distribution were characterized. A total of 115 up-regulated circRNAs and 146 down-regulated circRNAs in LC goats were screened compared to ZB goats. The authenticity of 10 differentially expressed circRNAs was validated by detecting their expression levels and the head-to-tail splice junction using RT-PCR and DNA sequencing, respectively. The parent genes of differentially expressed circRNA were mainly enriched in some Gene Ontology (GO) terms and pathways related to cashmere fiber traits, such as the canonical Wnt signaling pathway, which is involved in the regulation of cell promotion, stem cell proliferation, Wnt signaling pathway regulation, epithelial morphogenesis, MAPK signaling pathway, and cell adhesion molecules pathway. Eight differentially expressed circRNAs were further selected to construct a circRNA-miRNA network, and some miRNAs that were previously reported as related to fiber traits were found in the network. This study provides a deep understanding of the roles of circRNAs in the regulation of cashmere fiber traits in cashmere goats and the involvement of differential splicing in phenotypic expression according to breed and region.

RNA-Seq Reveals the Roles of Long Non-Coding RNAs (lncRNAs) in Cashmere Fiber Production Performance of Cashmere Goats in China.

Long non-coding RNAs (lncRNAs) are a kind of non-coding RNA being >200 nucleotides in length, and they are found to participate in hair follicle growth and development and wool fiber traits regulation. However, there are limited studies reporting the role of lncRNAs in cashmere fiber production in cashmere goats. In this study, Liaoning cashmere (LC) goats (n = 6) and Ziwuling black (ZB) goats (n = 6) with remarkable divergences in cashmere yield, cashmere fiber diameter, and cashmere color were selected for the construction of expression profiles of lncRNAs in skin tissue using RNA sequencing (RNA-seq). According to our previous report about the expression profiles of mRNAs originated from the same skin tissue as those used in the study, the cis and trans target genes of differentially expressed lncRNAs between the two caprine breeds were screened, resulting in a lncRNA-mRNA network. A total of 129 lncRNAs were differentially expressed in caprine skin tissue samples between LC goats and ZB goats. The presence of 2 cis target genes and 48 trans target genes for the differentially expressed lncRNAs resulted in 2 lncRNA-cis target gene pairs and 93 lncRNA-trans target gene pairs. The target genes concentrated on signaling pathways that were related to fiber follicle development, cashmere fiber diameter, and cashmere fiber color, including PPAR signaling pathway, metabolic pathways, fatty acid metabolism, fatty acid biosynthesis, tyrosine metabolism, and melanogenesis. A lncRNA-mRNA network revealed 22 lncRNA-trans target gene pairs for seven differentially expressed lncRNAs selected, of which 13 trans target genes contributed to regulation of cashmere fiber diameter, while nine trans target genes were responsible for cashmere fiber color. This study brings a clear explanation about the influences of lncRNAs over cashmere fiber traits in cashmere goats.

The Identification and Characteristics of miRNAs Related to Cashmere Fiber Traits in Skin Tissue of Cashmere Goats.

microRNAs (miRNAs) are involved in the regulation of biological phenomena by down-regulating the expression of mRNAs. In this study, Liaoning cashmere (LC) goats (n = 6) and Ziwuling black (ZB) goats (n = 6) with different cashmere fiber production performances were selected. We supposed that miRNAs are responsible for the cashmere fiber trait differences. To test the hypothesis, the expression profiles of miRNAs from the skin tissue of the two caprine breeds were compared using small RNA sequencing (RNA-seq). A total of 1293 miRNAs were expressed in the caprine skin samples, including 399 known caprine miRNAs, 691 known species-conserved miRNAs, and 203 novel miRNAs. Compared with ZB goats, 112 up-regulated miRNAs, and 32 down-regulated miRNAs were found in LC goats. The target genes of the differentially expressed miRNAs were remarkably concentrated on some terms and pathways associated with cashmere fiber performance, including binding, cell, cellular protein modification process, and Wnt, Notch, and MAPK signaling pathways. The miRNA-mRNA interaction network found that 14 miRNAs selected may contribute to cashmere fiber traits regulation by targeting functional genes associated with hair follicle activities. The results have reinforced others leading to a solid foundation for further investigation of the influences of individual miRNAs on cashmere fiber traits in cashmere goats.

Integrated transcriptome analysis reveals roles of long non-coding RNAs (lncRNAs) in caprine skeletal muscle mass and meat quality.

Long non-coding RNAs (lncRNAs) play important roles in the growth and development of skeletal muscle. However, there is limited information on goats. In this study, expression profiles of lncRNAs in Longissimus dorsi muscle from Liaoning cashmere (LC) goats and Ziwuling black (ZB) goats with divergent meat yield and meat quality were compared using RNA-sequencing. Based on our previous microRNA (miRNA) and mRNA profiles obtained from the same tissues, the target genes and binding miRNAs of differentially expressed lncRNAs were obtained. Subsequently, lncRNA-mRNA interaction networks and a ceRNA network of lncRNA-miRNA-mRNA were constructed. A total of 136 differentially expressed lncRNAs were identified between the two breeds. Fifteen cis target genes and 143 trans target genes were found for differentially expressed lncRNAs, and they were enriched in muscle contraction, muscle system process, muscle cell differentiation, and p53 signaling pathway. A total of 69 lncRNA-trans target gene pairs were constructed, with close relationship with muscle development, intramuscular fat deposition, and meat tenderness. A total of 16 lncRNA-miRNA-mRNA ceRNA pairs were identified, of which some reportedly associated with skeletal muscle development and fat deposition were found. The study will provide an improved understanding of the roles of lncRNAs in caprine meat yield and meat quality.

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.