TCEA3 promotes differentiation of C2C12 cells via an Annexin A1-mediated transforming growth factor-ß signaling pathway.

TCEA3 is a member of the transcription elongation factor family that not only promotes transcription but may also participate in other cytoplasmic processes. However, its mechanisms of action remain unclear. Our previous study indicated that TCEA3 may affect muscle differentiation. In this study, we investigated the expression and localization of TCEA3 in C2C12 cells and examined the role of TCEA3 in differentiation, its interaction with other cell proteins, and mechanisms of action. We found that the expression of TCEA3 increased gradually with an increase in the number of differentiation days and that it is mainly expressed in the cytoplasm of C2C12 cells, of which it promotes differentiation. Coimmunoprecipitation experiments and western blot analysis revealed that TCEA3 interacts with Annexin A1 (ANXA1), which is located in the cytoplasm and also promotes cell differentiation. Collectively, our results indicate that TCEA3 promotes cell differentiation by interacting with ANXA1 and affecting transforming growth factor-ß signaling pathways.

Platelet endothelial aggregation receptor-1 (PEAR1) is involved in C2C12 myoblast differentiation.

C2C12 murine myoblasts are a common model for studying muscle differentiation. Platelet endothelial aggregation receptor-1 (PEAR1), an epidermal growth factor repeat-containing transmembrane receptor, is known to participate in platelet contact-induced activation. In the present study, we demonstrated that PEAR1 is involved in the differentiation of C2C12 murine myoblasts. Western blotting and immunofluorescence staining were used to determine PEAR1 expression and localization during C2C12 cell differentiation. Subsequently, PEAR1 expression was activated and inhibited using clustered regularly interspaced short palindromic repeats-dCas9 technology to explore its effects on this process. PEAR1 expression was found to increase over the course of C2C12 cell differentiation. This protein was predominately localized on the membrane of these cells, where it clustered upon induction of differentiation. Expression of the myogenic markers Desmin, MYOG, and MYH2 revealed that PEAR1 positively regulated C2C12 cell differentiation. Moreover, induction of muscle injury by administration of bupivacaine to mice indicated that PEAR1 might play a role in muscle regeneration. In summary, our study confirmed the involvement of PEAR1 in C2C12 cell differentiation, contributing to our understanding of the molecular mechanisms underlying muscle development.

Effect of TCEA3 on the differentiation of bovine skeletal muscle satellite cells.

Bovine muscle-derived satellite cells (MDSCs) are important for animal growth. In this study, the effect of transcription elongation factor A3 (TCEA3) on bovine MDSC differentiation was investigated. Western blotting, immunofluorescence assays, and cytoplasmic and nuclear protein isolation and purification techniques were used to determine the expression pattern and protein localization of TCEA3 in bovine MDSCs during in vitro differentiation. TCEA3 expression was upregulated using the CRISPR/Cas9 technique to study its effects on MDSC differentiation in vitro. TCEA3 expression gradually increased during the in vitro differentiation of bovine MDSCs and peaked on the 5th day of differentiation. TCEA3 was mainly localized in the cytoplasm of bovine MDSCs, and its expression was not detected in the nucleus. The level of TCEA3 was relatively higher in myotubes at a higher degree of differentiation than during early differentiation. After transfection with a TCEA3-activating plasmid vector (TCEA3 overexpression) for 24 h, the myotube fusion rate, number of myotubes, and expression levels of the muscle differentiation-related loci myogenin (MYOG) and myosin heavy chain 3 (MYH3) increased significantly during the in vitro differentiation of bovine MDSCs. After transfection with a TCEA3-inhibiting plasmid vector for 24 h, the myotube fusion rate, number of myotubes, and expression levels of MYOG and MYH3 decreased significantly. Our results indicated, for the first time, that TCEA3 promotes the differentiation of bovine MDSCs and have implications for meat production and animal rearing.

Effect of ELOVL3 expression on bovine skeletal muscle-derived satellite cell differentiation.

ELOVL3 is involved in elongating saturated and monounsaturated fatty acids, and is a critical enzyme for lipid accumulation in brown adipocytes during the early phase of tissue recruitment. In addition, ELOVL3 is related to increased fatty acid oxidation in brown adipocytes. However, the potential functions of ELOVL3 in bovine cells remain unclear. Herein, we aimed to elucidate the effect of the ELOVL3 on the differentiation of bovine skeletal muscle-derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used for elucidating ELOVL3 expression pattern in bovine MDSCs during differentiation in vitro. We activated or inhibited ELOVL3 to study the effect of alterations in its expression on in vitro differentiation of bovine MDSCs. ELOVL3 expression increased gradually during bovine MDSC differentiation, and its levels were higher in the more highly differentiated myotubes. Activation of ELOVL3 promoted MDSC differentiation, while inhibition of ELOVL3 hindered differentiation of these cells. Here, for the first time, we demonstrate the importance of ELOVL3 during bovine MDSC differentiation, which may assist in increasing beef cattle muscularity.

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.

Effect of differentiation on microRNA expression in bovine skeletal muscle satellite cells by deep sequencing.

BACKGROUND: The differentiation of skeletal muscle-derived satellite cells (MDSCs) is important in controlling muscle growth, improving livestock muscle quality, and healing of muscle-related disease. MicroRNAs (miRNAs) are a class of gene expression regulatory factors, which play critical roles in the regulation of muscle cell differentiation. This study aimed to compare the expression profile of miRNAs in MDSC differentiation, and to investigate the miRNAs which are involved in MDSC differentiation. METHOD: Total RNA was extracted from MDSCs at three different stages of differentiation (MDSC-P, MDSC-D1 and MDSC-D3, representing 0, 1 and 3 days after differentiation, respectively), and used to construct small RNA libraries for RNA sequencing (RNA-seq). RESULTS: The results showed that in total 617 miRNAs, including 53 novel miRNA candidates, were identified. There were 9 up-expressed, 165 down-expressed, and 15 up-expressed, 145 down-expressed in MDSC-D1 and MDSC-D3, respectively, compared to those in MDSC-P. Also, 17 up-expressed, 55 down-expressed miRNAs were observed in MDSC-D3 compared to those in MDSC-D1. All known miRNAs belong to 237 miRNA gene families. Furthermore, we observed some sequence variants and base edits of the miRNAs. GO and KEGG pathway analysis showed that the majority of target genes regulated by miRNAs were involved in cellular metabolism, pathways in cancer, actin cytoskeleton regulation and the MAPK signaling pathway. Regarding the 53 novel miRNAs, there were 7 up-expressed, 31 down-expressed, and 8 up-expressed, 26 down-expressed in MDSC-D1 and MDSC-D3, respectively, compared to those in MDSC-P. The expression levels of 12 selected miRNA genes detected by RT-qPCR were consistent with those generated by deep sequencing. CONCLUSIONS: This study confirmed the authenticity of 564 known miRNAs and identified 53 novel miRNAs which were involved in MDSC differentiation. The identification of novel miRNAs has significantly expanded the repertoire of bovine miRNAs and could contribute to advances in understanding muscle development in cattle.

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.

Transcriptional profiling of bovine muscle-derived satellite cells during differentiation in vitro by high throughput RNA sequencing.

In this study, we utilized high throughput RNA sequencing to obtain a comprehensive gene expression profile of muscle-derived satellite cells (MDSCs) upon induction of differentiation. MDSCs were cultured in vitro and RNA was extracted for sequencing prior to differentiation (MDSC-P), and again during the early and late differentiation (MDSC-D1, and MDSC-D3, respectively) stages. Sequence tags were assembled and analyzed by digital gene expression profile to screen for differentially expressed genes, Gene Ontology annotation, and pathway enrichment analysis. Quantitative real-time PCR was used to confirm the results of RNA sequencing. Our results indicate that certain of genes were changed during skeletal muscle cell development, cell cycle progression, and cell metabolism during differentiation of bovine MDSCs. Furthermore, we identified certain genes that could be used as novel candidates for future research of muscle development. Additionally, the sequencing results indicated that lipid metabolism might be the predominant cellular process that occurs during MDSC differentiation.

Blinatumomab as bridging therapy in two children with B-cell acute lymphoblastic leukemia complicated by invasive fungal disease / 中国当代儿科杂志

This article reports two cases of children with B-cell acute lymphoblastic leukemia (B-ALL) complicated by invasive fungal disease (IFD) who received bridging treatment using blinatumomab. Case 1 was a 4-month-old female infant who experienced recurrent high fever and limb weakness during chemotherapy. Blood culture was negative, and next-generation sequencing (NGS) of peripheral blood, bronchoalveolar lavage fluid, and cerebrospinal fluid were all negative. Chest CT and cranial MRI revealed obvious infection foci. Case 2 was a 2-year-old male patient who experienced recurrent high fever with multiple inflammatory masses during chemotherapy. Candida tropicalis was detected in peripheral blood and abscess fluid using NGS, while blood culture and imaging examinations showed no obvious abnormalities. After antifungal and blinatumomab therapy, both cases showed significant improvement in symptoms, signs, and imaging, and B-ALL remained in continuous remission. The report indicates that bridging treatment with blinatumomab in children with B-ALL complicated by IFD can rebuild the immune system and control the underlying disease in the presence of immunosuppression and severe fungal infection.

MiR-2425-5p targets RAD9A and MYOG to regulate the proliferation and differentiation of bovine skeletal muscle-derived satellite cells.

Our group previously identified miR-2425-5p, a unique bovine miRNA; however, its biological function and regulation in muscle-derived satellite cells (MDSCs) remain unclear. Herein, stem-loop RT-PCR results showed that miR-2425-5p increased during MDSCs proliferation, but decreased during differentiation. Cell proliferation was examined using EdU assays, cyclin B1 (CCNB1) and proliferating cell nuclear antigen (PCNA) western blot (WB) and flow cytometry analysis. These results showed that miR-2425-5p mimics (miR-2425-M) enhanced MDSCs proliferation, whereas, miR-2425-5p inhibitor (miR-2425-I) had opposite effect. Conversely, cell differentiation studies by desmin (DES) immunofluorescence, myotubes formation, and myosin heavy chain 3 (MYH3) WB analyses revealed that miR-2425-M and miR-2425-I blocked and promoted MDSCs differentiation, respectively. Moreover, luciferase reporter, RT-PCR, and WB assays showed that miR-2425-5p directly targeted the 3'-UTR of RAD9 homolog A (RAD9A) and myogenin (MYOG) to regulate their expression. Rescue experiment showed RAD9A inhibited the proliferation of MDSCs through miR-2425-5p. In addition, we found that miR-2425-5p expression was regulated by its host gene NCK associated protein 5-like (NCKAP5L) rather than being transcribed independently as a separate small RNA. Collectively, these data indicate that miR-2425-5p is a novel regulator of bovine MDSCs proliferation and differentiation and provides further insight into the biological functions of miRNA in this species.

Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line.

To study milk synthesis in dairy goat mammary gland, we had established an in vitro lactating dairy goat mammary epithelial cell (DGMEC) line. Mammary tissues of Guan Zhong dairy goats at 35 d of lactation were dispersed and cultured in a medium containing epithelial growth factor, insulin-like growth factor-1, insulin transferrin serum, and fetal bovine serum. Epithelial cells were enriched by digesting with 0.25% trypsin repeatedly to remove fibroblast cells and were identified as epithelial origin by staining with antibody against cytokeratine 18. The DGMECs displayed monolayer, cobble-stone, epithelial-like morphology, and formed alveoli-like structures and island monolayer aggregates which were the typical characteristics of mammary epithelial cells. A one-half logarithmically growth curve and cytoplasmic lipid droplets in these cells were observed. In this paper, we also studied the lactating function of DGMECs. Results showed that DGMECs could secrete lactose and ß-casein. Lactating function of the cells had no obvious change after 48 h treated by insulin, while prolactin could obviously raise the secretion of milk proteins and lactose.

Stat5a increases lactation of dairy cow mammary gland epithelial cells cultured in vitro.

Signal transducer and activator of transcription 5a (Stat5a) transduces signals of extracellular cytokines and growth factors to the nucleus of mammary gland epithelial cells and thereby regulates gene transcription during pregnancy, lactation, and weaning. However, its function on the milk production of dairy cows needs further investigation. In this experiment, the effects of Stat5a on lactation ability of dairy cow mammary gland epithelial cells (DCMECs) were analyzed. Eukaryotic expression vector pcDNA3.1+-stat5a-αS1 was constructed by inserting stat5a gene into the plasmid vector pcDNA3.1+ and replacing CMV promoter with α-S1-casein 5' flanking sequence. The recombinant vector was stably transfected into DCMECs after geneticin (G418) selection. The proliferation and viability of DCMECs, expression of ß-casein and stat5a gene, and the content of lactose were detected. The results showed that stat5a gene in eukaryotic expression vector pcDNA3.1+-stat5a-αS1 was highly expressed in DCMECs and could increase the lactation ability of DCMECs. The associativity of Stat5a with nutrients on the lactation ability of DCMECs was also evaluated. Lysine (Lys), methionine (Met), sodium acetate, ß-sodium hydroxybutyrate, and glucose all had more positive effects on the lactation function of DCMECs after pcDNA3.1+-stat5a-αS1 transfection. The proliferation and viability of DCMECs, expression of ß-casein and stat5a gene, and contents of lactose and triglyceride were detected. The results revealed that nutrients could promote expression of Stat5a gene to increase lactation of DCMECs. These data help to clarify the function of stat5 gene on lactation and gene regulatory networks linking stat5a.