Yunqin Yan

Galactopoietic Activity of Dibutyl Phthalate Isolated from Vaccaria segetalis

Abstract A galactopoietic compound, identified as dibutyl phthalate (DBP), was isolated from Vaccaria segetalis . The activity of DBP on lactation ability of dairy cow mammary gland epithelial cells (DCMECs) cultured in vitro and dairy cow was evaluated. Results showed that DBP could promote cell viability, proliferation ability, lactose and β-casein secretion of DCMECs, which could also raise the milk yields of dairy cows significantly.

Transcription Factor EGR1 Promotes Differentiation of Bovine Skeletal Muscle Satellite Cells by Regulating MyoG Gene Expression.

The transcription factor, early growth response 1 (EGR1), has important roles in various cell types in response to different stimuli. EGR1 is thought to be involved in differentiation of bovine skeletal muscle‐derived satellite cells (MDSCs); however, the precise effects of EGR1 on differentiation of MDSCs and its mechanism of action remain unknown. In the present study, a time course of EGR1 expression and the effects of EGR1 on MDSC differentiation were determined. The results demonstrated that the expression of EGR1 mRNA and protein increased significantly in differentiating MDSCs relative to that in proliferating cells. Over‐expression of the EGR1 gene in MDSCs promoted their differentiation and inhibited proliferation. Conversely, knock‐down of EGR1 inhibited differentiation of MDSCs and promoted their proliferation, indicating that EGR1 promotes MDSC differentiation. Moreover, over‐expression of EGR1 in MDSCs increased the expression of MyoG mRNA and protein, whereas its knock‐down had the opposite effect. Furthermore, ChIP‐PCR analyses demonstrated that EGR1 could bind directly to its putative binding site within the promoter region of MyoG, and determination of ERG1 subcellular localization in MDSCs demonstrated that it could relocate to the nucleus, indicating MyoG is likely an EGR1 target gene whose expression is positively regulated by this transcription factor. In conclusion, EGR1 can promote MDSC differentiation through positive regulation of MyoG gene expression.

Fatty acids promote bovine skeletal muscle satellite cell differentiation by regulating ELOVL3 expression

Fatty acids (FAs) play essential roles in regulating differentiation and proliferation by affecting gene expression in various cell types. However, their potential functions in bovine cells remain unclear. Herein, we examine the differentiation and proliferation of bovine skeletal muscle-derived satellite cells (MDSCs) after incubation with three types of representative FAs (palmitic acid, oleic acid and docosahexaenoic acid) by western blotting, immunofluorescence assays, flow cytometry analysis and EdU incorporation assays. The myotube fusion rate, myotube length and expression levels of muscle differentiation-related gene myogenin (MYOG) and myosin heavy chain 3 (MYH3) increased significantly, although the FAs did not affect proliferation. Additionally, FA-induced bovine MDSC differentiation increased ELOVL3 expression and relocation of ELOVL3 to cytoplasmic lipid droplets in the differentiation of bovine MDSCs. Moreover, the effect of FAs on bovine MDSC differentiation was inhibited upon ELOVL3 downregulation. Collectively, these data indicate that FAs promote bovine MDSC differentiation by regulating ELOVL3 expression.

GGeneration of an efficient artificial promoter of bovine skeletal muscle alpha-actin gene (ACTA1) through addition of cis-acting element

Abstract The promoter of skeletal muscle α-actin gene (ACTA1) is highly muscle specific. The core of the bovine ACTA1 promoter extends from +29 to −233, about 262 base pairs (bp), which is sufficient to activate transcription in bovine muscle satellite cells. In this study, analysis by PCR site-specific mutagenesis showed that the cis-acting element SRE (serum response element binding factor) was processed as a transcriptional activator. In order to enhance the bovine ACTA1 promoter’s activity, we used a strategy to modify it. We cloned a fragment containing three SREs from the promoter of ACTA1, and then one or two clones were linked upstream of the core promoter (262 bp) of ACTA1. One and two clones increased the activity of the ACTA1 promoter 3-fold and 10-fold, respectively, and maintained muscle tissue specificity. The modified promoter with two clones could increase the level of ACTA1 mRNA and protein 4-fold and 1.1-fold, respectively. Immunofluorescence results showed that green fluorescence of ACTA1 increased. Additionally, the number of total muscle microfilaments increased. These genetically engineered promoters might be useful for regulating gene expression in muscle cells and improving muscle mass in livestock.

Effects of CMV Enhancer on Activity and Specificity of Bovine MyoG Gene Promoter

Abstract Connected a segment of CMV enhancer to the front of MyoG gene promoter and then constructed the corresponding dual luciferase expression vector pGL3-CMV-MyoGpro. We set four eukaryotic expression vectors including pGL3-CMV, pGL3-MyoGpro, pGL3-CMV-MyoGpro, and pGL3-Basic which contained CMV promoter, MyoG promoter, CMV-MyoG synthesis promoter, and a promoterless negative control, respectively. Then the four vectors and internal control Renilla luciferase report gene vector phRL-TK were transfected into bovine skeletal muscle satellite cells, mouse C2C12 cells and bovine fetal fibroblast cells to detect the promoter activity with dual luciferase report system. The results showed that CMV enhancer could significantly improve the transcription activity of bovine MyoG gene promoter in muscle satellite cells and mouse C2C12 cells, and it had certain specificity. This study provided experimental materials for increasing the high expression of exogenous gene in bovine muscle cells, and also laid the molecular theoretical basis for obtaining the high specific promoter of bovine muscle and the transgenic beef cattle.

MiR-139 promotes differentiation of bovine skeletal muscle-derived satellite cells by regulating DHFR gene expression: ZHOU et al.

MicroRNAs play an important regulatory role in the proliferation and differentiation of skeletal muscle‐derived satellite cells (MDSCs). In particular, miR‐139 can inhibit tumor cell proliferation and invasion, and its expression is down‐regulated during C2C12 myoblast differentiation. The aim of this study was thus to examine the effect and potential mechanism of miR‐139 in bovine MDSCs. The expression of miR‐139 was found to be significantly increased during bovine MDSC differentiation by stem‐loop reverse transcription‐polymerase chain reaction amplification. Statistical analysis of the myotube fusion rate was done through immunofluorescence detection of desmin, and western blotting was used to measure the change in protein expression of the muscle differentiation marker genes MYOG and MYH3. The results showed that the miR‐139 mimic could enhance the differentiation of bovine MDSCs, whereas the inhibitor had the opposite effect. By using the dual‐luciferase reporter system, miR‐139 was found to target the 3′‐untranslated region of the dihydrofolate reductase (DHFR) gene and regulate its expression. In addition, the expression of miR‐139 was found to be regulated by its host gene phosphodiesterase 2A (PDE2A) via inhibition of the latter by CRISPR interference (CRISPRi). Overall, our findings indicate that miR‐139 plays an important role in regulating the differentiation of bovine MDSCs.

Effect of ECM2 expression on bovine skeletal muscle-derived satellite cell differentiation: ECM2 promotes satellite cell differentiation

Extracellular matrix components have important regulatory functions during cell proliferation and differentiation. In recent study, extracellular matrix were shown to have a strong effect on skeletal muscle differentiation. Here, we aimed to elucidate the effects of extracellular matrix protein 2 (ECM2), an extracellular matrix component, on the differentiation of bovine skeletal muscle‐derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used to elucidate the ECM2 expression pattern in bovine MDSCs during differentiation in vitro. CRISPR/Cas9 technology was used to activate or inhibit ECM2 expression to study its effects on the in vitro differentiation of bovine MDSCs. ECM2 expression was shown to increase gradually during bovine MDSC differentiation, and the levels of this protein were higher in more highly differentiated myotubes. ECM2 activation promoted MDSC differentiation, whereas its suppression inhibited the differentiation of these cells. Here, for the first time, we demonstrated the importance of ECM2 expression during bovine MDSC differentiation; these results could lead to treatments that help to increase beef cattle muscularity.

Effects of COL8A1 on the proliferation of muscle-derived satellite cells: COL8A1 promotes bovine MDSC proliferation

Collagen type VIII alpha 1 chain (COL8A1) is a component of the extracellular matrix. Our previous studies suggested that COL8A1 is associated with the proliferation of muscle‐derived satellite cells (MDSCs). Additionally, it has been demonstrated that COL8A1 promotes the proliferation of smooth muscle cells and liver cancer cells. Therefore, we predicted that COL8A1 is associated with the proliferation of bovine MDSCs, which have potential applications in research. In this study, we constructed vectors to activate and repress COL8A1 in bovine MDSCs using the CRISPR/Cas9 technique and determined the effects of COL8A1 modulation by EdU labeling, Western blotting, and dual‐luciferase reporter assays. The results showed that activation of COL8A1 increased the number of EdU‐positive cells and expression of the proliferation markers cyclin B1 (CCNB1) and P‐AKT. The expression of P‐Akt was unchanged after addition of LY294002 (a protein kinase inhibitor capable of blocking the signal transduction pathway of the phosphoinositide 3‐kinase). In contrast, repression of COL8A1 reduced the number of EdU‐positive cells and expression of CCNB1 and P‐AKT. We also observed upregulation and downregulation of COL8A1 following the overexpression and repression of EGR1, respectively. The dual‐luciferase reporter assay revealed that EGR1 regulates the promoter activity of COL8A1. To our knowledge, this is the first study demonstrating that EGR1 positively regulates the expression of COL8A1, which in turn promotes the proliferation of bovine MDSCs via the PI3 K/AKT signaling pathway.