CircSSBP2 acts as a MiR-2400 sponge to promote intramuscular preadipocyte proliferation by regulating NDRG1.

Intramuscular fat (IMF) is a critical factor in beef quality. IMF is mainly distributed between muscle fibres and its accumulation can affect the marbling and meat quality of beef. IMF formation and deposition is a complex process and in recent years a group of non-coding RNAs (ncRNAs), known as circRNAs, have been discovered to play an important role in regulating intramuscular fat deposition. CircRNAs form a covalent loop structure after reverse splicing of precursor mRNAs. They can act by adsorbing miRNAs, thereby reducing their repressive effects on downstream target genes. Based on high-throughput sequencing of circRNAs in intramuscular fat of Qinchuan and Japanese black cattle, we identified a novel circSSBP2 that is differentially expressed between the two species and associated with adipogenesis. We show that circSSBP2 knockdown promotes bovine intramuscular preadipocyte proliferation, whereas overexpression inhibits bovine intramuscular preadipocyte proliferation. We also show that circSSBP2 can act as a molecular sponge for miR-2400 and that miR-2400 overexpression promotes bovine intramuscular preadipocyte proliferation. Furthermore, N-myc downstream-regulated gene 1 (NDRG1) was identified as a direct target gene of miR-2400, and NDRG1 interference promoted the proliferation of bovine intramuscular preadipocytes. In conclusion, our results suggest that circSSBP2 inhibits the proliferation of bovine intramuscular preadipocytes by regulating the miR-2400/NDRG1 axis.

IFRD2, a target of miR-2400, regulates myogenic differentiation of bovine skeletal muscle satellite cells via decreased phosphorylation of ERK1/2 proteins.

The proliferation and differentiation of skeletal muscle satellite cells is a complex physiological process involving various transcription factors and small RNA molecules. This study aimed to understand the regulatory mechanisms underlying these processes, focusing on interferon-related development factor 2 (IFRD2) as a target gene of miRNA-2400 in bovine skeletal MuSCs (MuSCs). IFRD2 was identified as a target gene of miRNA-2400 involved in regulating the proliferation and differentiation of bovine skeletal MuSCs. Our results indicate that miR-2400 can target binding the 3'UTR of IFRD2 and inhibit its translation. mRNA and protein expression levels of IFRD2 increased significantly with increasing days of differentiation. Moreover, overexpression of the IFRD2 gene inhibited proliferation and promoted differentiation of bovine MuSCs. Conversely, the knockdown of the gene had the opposite effect. Overexpression of IFRD2 resulted in the inhibition of ERK1/2 phosphorylation levels in bovine MuSCs, which in turn promoted differentiation. In summary, IFRD2, as a target gene of miR-2400, crucially affects bovine skeletal muscle proliferation and differentiation by precisely regulating ERK1/2 phosphorylation.

MicroRNA-2400 promotes bovine preadipocyte proliferation.

MicroRNAs (miRNAs) play critical roles in the proliferation of bovine preadipocytes. miR-2400 is a novel and unique miRNA from bovines. In the present study, we separated and identified preadipocytes from bovine samples. miR-2400 overexpression increased the rate of preadipocyte proliferation, which was analyzed with a combination of EdU and flow cytometry. Simultaneously, functional genes related to proliferation (PCNA, CCND2, CCNB1) were also increased, which was detected by real-time PCR. Furthermore, luciferase reporter assays showed that miR-2400 bound directly to the 3'untranslated regions (3'UTRs) of PRDM11 mRNA. These data suggested that miR-2400 could promote preadipocyte proliferation by targeting PRDM11.

Identification of miR-2400 gene as a novel regulator in skeletal muscle satellite cells proliferation by targeting MYOG gene.

MicroRNAs play critical roles in skeletal muscle development as well as in regulation of muscle cell proliferation and differentiation. Previous study in our laboratory showed that the expression level of miR-2400, a novel and unique miRNA from bovine, had significantly changed in skeletal muscle-derived satellite cells (MDSCs) during differentiation, however, the function and expression pattern for miR-2400 in MDSCs has not been fully understood. In this report, we firstly identified that the expression levels of miR-2400 were down-regulated during MDSCs differentiation by stem-loop RT-PCR. Over-expression and inhibition studies demonstrated that miR-2400 promoted MDSCs proliferation by EdU (5-ethynyl-2' deoxyuridine) incorporation assay and immunofluorescence staining of Proliferating cell nuclear antigen (PCNA). Luciferase reporter assays showed that miR-2400 directly targeted the 3' untranslated regions (UTRs) of myogenin (MYOG) mRNA. These data suggested that miR-2400 could promote MDSCs proliferation through targeting MYOG. Furthermore, we found that miR-2400, which was located within the eighth intron of the Wolf-Hirschhorn syndrome candidate 1-like 1 (WHSC1L1) gene, was down-regulated in MDSCs in a direct correlation with the WHSC1L1 transcript by Clustered regularly interspaced palindromic repeats interference (CRISPRi). In addition, these observations not only provided supporting evidence for the codependent expression of intronic miRNAs and their host genes in vitro, but also gave insight into the role of miR-2400 in MDSCs proliferation.

Bta-miR-2400 Targets SUMO1 to Affect Yak Preadipocytes Proliferation and Differentiation.

Yak adipose tissue may have evolved a unique energy metabolism manner to accommodate the organism's seasonal growth rhythms. MiRNAs regulate multiple biological processes including systemic metabolism and energy homeostasis through post-transcriptional regulations. Rare reports have shown that miRNAs regulate lipid metabolism in domestic yaks. Therefore, we investigated the regulatory mechanisms of bta-miR-2400 in modulating yak preadipocytes proliferation and differentiation. We found that bta-miR-2400 was highly expressed in adipose tissue. Overexpression of bta-miR-2400 in yak preadipocytes significantly enhanced cell proliferation, increased the number of EdU fluorescence-stained cells, and promoted the expression of proliferation marker genes (CDK2, CDK4 and PCNA). Besides, overexpression of bta-miR-2400 repressed the expression of adipogenesis-related marker genes, and the content of cellular triglyceride was substantially reduced. Conversely, inhibition of bta-miR-2400 showed opposite effects compared to those of bta-miR-2400 overexpression in yak preadipocytes. Further, luciferase reporter assays revealed that SUMO1 is a target gene of bta-miR-2400, with bta-miR-2400 being able to down-regulate SUMO1 mRNA and protein expression. In conclusion, bta-miR-2400 regulates lipid metabolism and energy homeostasis in yak preadipocytes by directly targeting SUMO1 to promote cell proliferation and inhibit differentiation.

Circular RNA circMYL1 Inhibit Proliferation and Promote Differentiation of Myoblasts by Sponging miR-2400.

Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs (ncRNAs) involved in regulating skeletal muscle development by sponging miRNAs. In this study, we found that the circMYL1 expression was down-regulated during myoblast proliferation, while gradually up-regulated in myoblast differentiation. The potential role of circMYL1 was identified in the proliferation of bovine myoblast through mRNA and protein expression of proliferation marker genes (PCNA, CyclinD1, and CDK2), cell counting kit-8 assay, flow cytometry analysis, and 5-ethynyl 2'-deoxyuridine (EdU) assay. Analysis of the expression of differentiation marker genes (MyoD, MyoG, and MYH2) and immunofluorescence of Myosin heavy chain (MyHC) was used to assess cell differentiation. The proliferation analysis revealed that circMYL1 inhibited the proliferation of bovine primary myoblast. Furthermore, the differentiation analysis demonstrated that circMYL1 promoted the differentiation of bovine primary myoblast. The luciferase screening and RNA immunoprecipitation (RIP) assays found that circMYL1 could have interaction with miR-2400. Additionally, we demonstrated that miR-2400 promoted proliferation and inhibited differentiation of bovine primary myoblast, while circMYL1 may eliminate the effects of miR-2400, as showed by rescue experiments. Together, our results revealed that a novel circular RNA of circMYL1 could inhibit proliferation and promote differentiation of myoblast by sponging miR-2400.