MicroRNAs (miRNAs) have been found to be involved in lipid deposition and metabolism. However, there have been no reports on the roles of miR-148a in the proliferation and adipogenesis of preadipocytes in sheep. In this study, the expression of miR-148a was profiled in the eight tissues of Tibetan ewes and differentiated preadipocytes, and the role of miR-148a in differentiation and proliferation of ovine preadipocytes was investigated using Oil Red O staining, CCK-8, EdU staining, cell cycle detection, and RT-qPCR. The effect of PTEN on the differentiation of ovine preadipocytes was also investigated. The miR-148a was widely expressed in the eight tissues investigated and had significantly increased expression in liver, spleen and subcutaneous adipose tissues, and the heart. The expression of miR-148a continued to increase with the differentiation of ovine preadipocytes. The over-expression of miR-148a significantly promoted differentiation but inhibited the proliferation of ovine preadipocytes. The inhibition of miR-148a had the opposite effect on the differentiation and proliferation of ovine preadipocytes with over-expressed miR-148a. The results from the dual luciferase reporter assays showed that miR-148a mimic significantly decreased the luciferase activity of PTEN-3′UTR dual luciferase reporter vector, suggesting that PTEN is a target gene of miR-148a. In over-expressed-PTEN preadipocytes, the number of lipid droplets remarkably decreased, and the expression levels of adipogenesis marker genes PPARγ, FASN, FATP4, GLUT4, C/EBPβ and LPL were also significantly down-regulated. These results suggest that miR-148a accelerated the adipogenic differentiation of ovine preadipocytes by inhibiting PTEN expression, and also inhibited the proliferation of ovine preadipocytes.

Variation in some caprine keratin-associated protein (KAP) genes has been associated with cashmere fiber traits, but many KAP genes remain unidentified in goats. In this study, we confirm the identification of a KAP27-1 gene (KRTAP27-1) and describe its effect on cashmere traits in 248 Longdong cashmere goats. A polymerase chain reaction–single strand conformation polymorphism (PCR-SSCP) analysis was used to screen for sequence variation in this gene, and three sequence variants (named A to C) were found. These sequences have the highest similarity (77% identity) to a human KRTAP27-1 sequence, while sharing some homology with a predicted caprine KRTAP27-1 sequence ENSCHIG00000023347 in the goat genome construct (ARS1:CM004562.1) at chromosome 1 position 3,966,193–3,973,677 in the forward strand. There were two single nucleotide polymorphisms (SNPs) detected in the coding sequence, including one nonsynonymous SNP (c.413C/T; p.Ala138Val) and one synonymous SNP (c.495C/T). The C variant differed from A and B at c.413C/T, having cytosine in its nucleotide sequence, while the B variant differed from A and C at c.495C/T, having thymine in its nucleotide sequence. Goats of the genotypes AB and BB produced cashmere fibers of higher mean fiber diameter (MFD) than goats of genotype AA, but no difference in MFD was detected between the AB and BB goats. These results suggest that B is associated with increased MFD. Expression of the caprine KRTAP27-1 sequence was predominantly detected in the skin tissue of goats but not or only weakly detected in other tissues, including longissimus dorsi muscle, heart, kidney, liver, lung and spleen.

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.

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.

Long non-coding RNAs (lncRNAs) are a kind of non-coding RNA with >200 nucleotides in length. Some lncRNAs have been proven to have clear regulatory functions in many biological processes of mammals. However, there have been no reports on the roles of lncRNAs in ovine mammary gland tissues. In the study, the expression profiles of lncRNAs were studied using RNA-Seq in mammary gland tissues from lactating Small-Tailed Han (STH) ewes and Gansu Alpine Merino (GAM) ewes with different milk yield and ingredients. A total of 1894 lncRNAs were found to be expressed. Compared with the GAM ewes, the expression levels of 31 lncRNAs were significantly up-regulated in the mammary gland tissues of STH ewes, while 37 lncRNAs were remarkably down-regulated. Gene Ontogeny (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the target genes of differentially expressed lncRNAs were enriched in the development and proliferation of mammary epithelial cells, morphogenesis of mammary gland, ErbB signaling pathway, and Wnt signaling pathway. Some miRNA sponges of differentially expressed lncRNAs, reported to be associated with lactation and mammary gland morphogenesis, were found in a lncRNA-miRNA network. This study reveals comprehensive lncRNAs expression profiles in ovine mammary gland tissues, thereby providing a further understanding of the functions of lncRNAs in the lactation and mammary gland development of sheep.

In this study, the expression profiles of miR-148a were constructed in eight different ovine tissues, including mammary gland tissue, during six different developmental periods. The effect of miR-148a on the viability, proliferation, and milk fat synthesis of ovine mammary epithelial cells (OMECs) was investigated, and the target relationship of miR-148a with two predicted target genes was verified. The expression of miR-148a exhibited obvious tissue-specific and temporal-specific patterns. miR-148a was expressed in all eight ovine tissues investigated, with the highest expression level in mammary gland tissue (p < 0.05). Additionally, miR-148a was expressed in ovine mammary gland tissue during each of the six developmental periods studied, with its highest level at peak lactation (p < 0.05). The overexpression of miR-148a increased the viability of OMECs, the number and percentage of Edu-labeled positive OMECs, and the expression levels of two cell-proliferation marker genes. miR-148a also increased the percentage of OMECs in the S phase. In contrast, transfection with an miR-148a inhibitor produced the opposite effect compared to the miR-148a mimic. These results indicate that miR-148a promotes the viability and proliferation of OMECs in Small-tailed Han sheep. The miR-148a mimic increased the triglyceride content by 37.78% (p < 0.01) and the expression levels of three milk fat synthesis marker genes in OMECs. However, the miR-148a inhibitor reduced the triglyceride level by 87.11% (p < 0.01). These results suggest that miR-148a promotes milk fat synthesis in OMECs. The dual-luciferase reporter assay showed that miR-148a reduced the luciferase activities of DNA methyltransferase 1 (DNMT1) and peroxisome proliferator-activated receptor gamma coactivator 1-A (PPARGC1A) in wild-type vectors, suggesting that they are target genes of miR-148a. The expression of miR-148a was highly negatively correlated with PPARGC1A (r = −0.789, p < 0.001) in ovine mammary gland tissue, while it had a moderate negative correlation with DNMT1 (r = −0.515, p = 0.029). This is the first study to reveal the molecular mechanisms of miR-148a underlying the viability, proliferation, and milk fat synthesis of OMECs in sheep.