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.

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.

MiRNAs regulate follicle development and atresia, steroid production, granulosa cell (GC) proliferation, and apoptosis. However, the target genes and the functioning of novel miRNAs remain unexplored. We reveal the targeting relationship between novel-m0297-5p and WNT5A and the specific regulatory mechanism of GC proliferation in small-tailed Han sheep using whole transcriptomic sequencing. We performed whole transcriptomic sequencing on small-tailed Han sheep ovarian GCs supplemented with 10 ng/mL of transforming growth factor-β1 (TGF-β1) during the early stages. This led to identifying the differential expression of novel-m0297-5p and Wnt family member 5A (WNT5A) and predicting their targeting relationship. Based on this, we hypothesized that TGF-β1 could mediate novel-m0297-5p targeting WNT5A to participate in the proliferation process of GCs in small-tailed sheep. We confirmed the relationship between TGF-β1 and both novel-m0297-5p and WNT5A. The mimicry of novel-m0297-5p inhibited GC activity and proliferation. However, the inhibition of novel-m0297-5p yielded the opposite effect. We validated the binding site for novel m0297-5p within the 3′UTR of WNT5A using dual-luciferase reporter gene. TGF-β1 alleviated the impact induced by the mimicry of novel-m0297-5p on cell viability. Inhibitor co-transfection for both novel-m0297-5p and si-WNT5A suppressed the granulocyte proliferation induced by novel-m0297-5p inhibition. These findings suggest that TGF-β1 can mediate the inhibitory effect of novel-m0297-5p targeting WNT5A on GC proliferation and activity in small-tailed Han sheep. This study provides an experimental basis for research on the biological function of GCs and their impact on follicle development.

The proliferation and differentiation of skeletal muscle satellite cells (SMSCs) are responsible for the development of skeletal muscle. In our previous study, circ_002156 was found to be highly expressed in caprine Longissimus Dorsi muscle, but the regulatory role of the circular RNAs (circRNA) in goat SMSCs remains unclear. In this study, the authenticity of circ_002156 was validated, and its structurally characteristic and cellular localization as well as tissue expression of circ_002156 and its parent genes were investigated. Moreover, the effects of circ_002156 on the viability, proliferation, and differentiation of SMSCs were also studied. The circ_002156 is located on caprine chromosome 19 with a length of 36,478. The circRNA structurally originates from myosin heavy chain 2 (MYH2), MYH1, and MYH4 as well as intergenic sequences among the parent genes. RT-PCR and Sanger sequencing confirmed the authenticity of circ_002156. Most circ_002156 (55.5%) was expressed in the nuclei of SMSCs, while 44.5% of circ_002156 was located in the cytoplasm. The circ_002156 and its three parent genes had higher expression levels in the triceps brachii, quadriceps femoris, and longissimus dorsi muscle tissues than in the other five tissues. The expression of circ_002156 and its parent genes MYH1 and MYH4 reached the maximum on day 8 of differentiation, while MYH2 in expression reached the peak on day 4 after differentiation. The Pearson correlation coefficients revealed that circ_002156 had moderate or high positive correlations with the three parent genes in the expression of both quadriceps femoris muscle and SMSCs during different differentiation stages. The small interfering RNA circ_002156 (named si-circ_002156) remarkably increased the viability of the SMSCs. The si-circ_002156 also increased the number and parentage of Edu-labeled positive SMSCs as well as the expression levels of four cell proliferation marker genes. These suggest that circ_002156 inhibited the proliferation of SMSCs. Meanwhile, si-circ_002156 decreased the area of MyHC-labeled positive myotubes, the myotube fusion index, and myotube size as well as the expression of its three parent genes and four cell differentiation marker genes, suggesting a positive effect of circ_002156 on the differentiation of SMSCs. This study contributes to a better understanding of the roles of circ_002156 in the proliferation and differentiation of SMSCs.