Background: Adipocyte, the main cellular component of white adipose tissue, plays a vital role in energy balance in higher eukaryotes. In recent years, adipocytes have also been identified as a major endocrine organ involved in immunological responses, vascular diseases, and appetite regulation. In farm animals, fat content and categories are closely correlated with meat quality. MicroRNAs (miRNAs), a class of endogenous single-stranded non-coding RNA molecules, participate in the regulation of adipocyte differentiation and adipogenesis through regulating the transcription or translation of target mRNAs. MiR-378 plays an important role in a number of biological processes, including cell growth, cell differentiation, tumor cell survival and angiogenesis. Methods: In the present study, bioinformatics analysis and dual-luciferase reporter assay were used to identify and validate the target genes of miR-378. In vitro cell transfection, quantitative reverse transcription polymerase chain reaction (RT-qPCR), western blot analysis, Oil Red O staining, and triglyceride content measurement were conducted to analyze the effects of miR-378 on bovine preadipocyte differentiation. Results: MiR-378 was induced during adipocyte differentiation. In the differentiated adipocytes overexpressing miR-378, the volume of lipid droplets was enlarged, and the triglyceride content was increased. Moreover, the mRNA expression levels of the adipocyte differentiation marker genes, peroxisome proliferator-activated receptor gamma (PPARγ) and sterol regulatory element-binding protein (SREBP), were significantly elevated in the differentiated, mature adipocytes. In contrast, the mRNA expression level of preadipocyte factor 1 (Pref-1) was markedly reduced. E2F transcription factor 2 (E2F2) and Ras-related nuclear (RAN)-binding protein 10 (RANBP10) were the two target genes of miR-378. The mRNA expression levels of E2F2 and RANBP10 did not significantly change in bovine preadipocytes overexpressing miR-378. However, the protein expression levels of E2F2 and RANBP10 were markedly reduced. Conclusion: MiR-378 promoted the differentiation of bovine preadipocytes. E2F2 and RANBP10 were the two target genes of miR-378, and might involve in the effects of miR-378 on the bovine preadipocyte differentiation.

Background/Aims: Bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which are secreted by oocytes, are important regulators of follicular growth and development and ovarian function. These two factors can regulate the proliferation and apoptosis of cumulus cells via modulation of the Smad signaling pathway. Studies have shown that BMP15 and GDF9 can affect the level of miR-375, whereas the target gene of miR-375 is BMPR2, the type II receptor of BMP15 and GDF9. However, whether or how the BMP15/ GDF9-miR-375-BMPR2 pathway affects the proliferation and apoptosis of bovine cumulus cells through regulation of the Smad signaling pathway remains unclear. Methods: In this study, cumulus cells were first obtained from cumulus-oocyte complexes (COCs). Appropriate concentrations of BMP15 and GDF9 were added during the in vitro culture process. Cell Counting Kit-8 (CCK-8) analyses and flow cytometry were used to determine the effects of BMP15/GDF9 on bovine cumulus cells proliferation and apoptosis. Subsequently, miR-375 mimics, miR-375 inhibitor and BMPR2 siRNA were synthesized and used for transfection experiments. Western Blot analysis was used to detect changes before and after transfection in the expression levels of the BMP15/GDF9 type I receptors ALK4, ALK5 and ALK6; the phosphorylation levels of Smad2/3 and Smad1/5/8, which are key signaling pathway proteins downstream of BMP15/GDF9; the expression levels of PTX3, HAS2 and PTGS2, which are key genes involved in cumulus cells proliferation; and Bcl2/Bax, which are genes involved in apoptosis. Results: The addition of 100 ng/mL BMP15 or 200 ng/mL GDF9 or the combined addition of 50 ng/mL BMP15 and 100 ng/mL GDF9 effectively inhibited bovine cumulus cell apoptosis and promoted cell proliferation. BMP15/GDF9 negatively regulated miR-375 expression and positively regulated BMPR2 expression. High levels of miR-375 and inhibition of BMPR2 resulted in increased expression of ALK4 and decreased expression of PTX3, HAS2 and PTGS2, whereas miR-375 inhibition resulted in the opposite results. BMP15 and GDF9 significantly activated the levels of p-Smad2/3 and p-Smad1/5/8, whereas miR-375 inhibited the levels of p-Smad2/3 and p-Smad1/5/8 by negatively regulating BMPR2 and also led to apoptosis. Conclusion: BMP15 and GDF9 have synergistic effects and can act through miR-375 to affect the expression levels of type I receptor ALK4 and type II receptor BMPR2 and the activation of Smad signaling pathway, which subsequently affected the proliferation, spread and apoptosis of cumulus cells.

The differentiation of preadipocytes into adipose tissues is tightly regulated by various factors including miRNAs and cytokines. In this study, taking advantage of isolated porcine primary preadipocytes, we showed that ectopic expression of miR‐375 could change preadipocyte differentiation. In addition, bone morphogenetic protein receptor 2 (BMPR2) was identified as a direct target of miR‐375. Silencing BMPR2 had the same inhibition effects as overexpressing miR‐375 on the preadipocyte differentiation. Together, we demonstrated that miR‐375 is a negative regulator of adipogenic differentiation using porcine primary preadipocytes. These results clarified the role of miR‐375 in ex vivo adipogenic differentiation.