Smritee Pokharel

Identification of Genes Differentially Expressed in Myogenin Knock-Down Bovine Muscle Satellite Cells during Differentiation through RNA Sequencing Analysis

Background The expression of myogenic regulatory factors (MRFs) consisting of MyoD, Myf5, myogenin (MyoG) and MRF4 characterizes various phases of skeletal muscle development including myoblast proliferation, cell-cycle exit, cell fusion and the maturation of myotubes to form myofibers. Although it is well known that the function of MyoG cannot be compensated for other MRFs, the molecular mechanism by which MyoG controls muscle cell differentiation is still unclear. Therefore, in this study, RNA-Seq technology was applied to profile changes in gene expression in response to MyoG knock-down (MyoGkd) in primary bovine muscle satellite cells (MSCs). Results About 61–64% of the reads of over 42 million total reads were mapped to more than 13,000 genes in the reference bovine genome. RNA-Seq analysis identified 8,469 unique genes that were differentially expressed in MyoGkd. Among these genes, 230 were up-regulated and 224 were down-regulated by at least four-fold. DAVID Functional Annotation Cluster (FAC) and pathway analysis of all up- and down-regulated genes identified overrepresentation for cell cycle and division, DNA replication, mitosis, organelle lumen, nucleoplasm and cytosol, phosphate metabolic process, phosphoprotein phosphatase activity, cytoskeleton and cell morphogenesis, signifying the functional implication of these processes and pathways during skeletal muscle development. The RNA-Seq data was validated by real time RT-PCR analysis for eight out of ten genes as well as five marker genes investigated. Conclusions This study is the first RNA-Seq based gene expression analysis of MyoGkd undertaken in primary bovine MSCs. Computational analysis of the differentially expressed genes has identified the significance of genes such as SAP30-like (SAP30L), Protein lyl-1 (LYL1), various matrix metalloproteinases, and several glycogenes in myogenesis. The results of the present study widen our knowledge of the molecular basis of skeletal muscle development and reveal the vital regulatory role of MyoG in retaining muscle cell differentiation.

Depot-specific gene expression profiles during differentiation and transdifferentiation of bovine muscle satellite cells, and differentiation of preadipocytes

We report a systematic study of gene expression during myogenesis and transdifferentiation in four bovine muscle tissues and of adipogenesis in three bovine fat tissues using DNA microarray analysis. One hundred hybridizations were performed and 7245 genes of known and unknown function were identified as being differentially expressed. Supervised hierarchical cluster analysis of gene expression patterns revealed the tissue specificity of genes. A close relationship in global gene expression observed for adipocyte-like cells derived from muscle and adipocytes derived from intramuscular fat suggests a common origin for these cells. The role of transthyretin in myogenesis is a novel finding. Different genes were highly induced during the transdifferentiation of myogenic satellite cells and in the adipogenesis of preadipocytes, indicating the involvement of different molecular mechanisms in these processes. Induction of CD36 and FABP4 expression in adipocyte-like cells and adipocytes may share a common pathway.

Synthesis, characterization and anticancer screening of some novel piperonyl-tetrazole derivatives.

A series of new 1,2-substituted tetrazole derivatives were synthesized and evaluated on MCF-7 (ER positive), MDA-MB-231 and ZR-75 (ER negative) breast cancer cell lines. Out of the fourteen compounds, three compounds 10, 12 and 14 showed higher inhibitory effects on MCF-7 cells. Whereas, compound 8 exhibited higher inhibition in MDA-MB-231 and ZR-75 cells at 10(-5) M concentration. Total RNA was extracted and effect of compounds on different marker genes was studied. The gene expression of CD44, BRAC and BAX were significantly affected. The compounds were screened against the HepG2 cell line, to know if they are selectively targeting specific cancers and only 1-10 percent inhibition was found at 10(-5) M concentration.

Transthyretin is a key regulator of myoblast differentiation.

Transthyretin (TTR) is a known carrier protein for thyroxine (T4) and retinol-binding protein in the blood that is primarily synthesized in the liver and choroid plexus of the brain. Herein, we report that the TTR gene is expressed in skeletal muscle tissue and up-regulated during myotube formation in C2C12 cells. TTR silencing (TTRkd) significantly reduced myogenin expression and myotube formation, whereas myogenin silencing (MYOGkd) did not have any effect on TTR gene expression. Both TTRkd and MYOGkd led to a decrease in calcium channel related genes including Cav1.1, STIM1 and Orai1. A significant decrease in intracellular T4 uptake during myogenesis was observed in TTRkd cells. Taken together, the results of this study suggest that TTR initiates myoblast differentiation via affecting expression of the genes involved during early stage of myogenesis and the genes related to calcium channel.

Expressed Sequence Tags for Bovine Muscle Satellite Cells, Myotube Formed-Cells and Adipocyte-Like Cells

Background Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs. Results A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay. Conclusion In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.

Characterization of gender-specific bovine serum

Animal cell cultures generally require a nutrient-rich medium supplemented with animal serum. Adult bovine serum contains a variety of nutrients including inorganic minerals, vitamins, salts, proteins and lipids as well as growth factors that promote animal cell growth. To evaluate the potential use of gender-specific bovine serum (GSBS) for cell culture, the biochemical properties of male serum (MS), female serum (FS) and castrated-male serum (CMS) were investigated. Overall, the chemical profile of GSBS was similar to that of bovine references except for glucose, creatine kinase, lactate dehydrogenase and potassium. FS showed elevated total protein and sodium concentrations compared to MS and CMS. Proteins present in MS, FS and CMS but absent in fetal bovine serum (FBS) were selected by two-dimensional gel electrophoresis and identified by peptide mass fingerprinting. Some of the identified proteins are known to be involved in immune responses and the others have unknown physiological roles. Moreover, it was found that some proteins such as alpha-2-macroglobulin appeared to be gender-specific with higher contents in FS. Insulin and testosterone was significantly higher in MS, and 17β-estradiol and estrone were higher in FS, as compared to the other sera. Taken together, the results indicate that each GSBS has a different ratio of components. Differences in serum constituents may affect cell cultures in a different manner and could be beneficial, depending on the specific aim of cell cultures.

Expressional studies of the aldehyde oxidase (AOX1) gene during myogenic differentiation in C2C12 cells

Aldehyde oxidases (AOXs), which catalyze the hydroxylation of heterocycles and oxidation of a wide variety of aldehydic compounds, have been present throughout evolution from bacteria to humans. While humans have only a single functional aldehyde oxidase (AOX1) gene, rodents are endowed with four AOXs; AOX1 and three aldehyde oxidase homologs (AOH1, AOH2 and AOH3). In continuation of our previous study conducted to identify genes differentially expressed during myogenesis using a microarray approach, we investigated AOX1 with respect to its role in myogenesis to conceptualize how it is regulated in C2C12 cells. The results obtained were validated by silencing of the AOX1 gene. Analysis of their fusion index revealed that formation of myotubes showed a marked reduction of up to 40% in AOX1kd cells. Expression of myogenin (MYOG), one of the marker genes used to study myogenesis, was also found to be reduced in AOX1kd cells. AOX1 is an enzyme of pharmacological and toxicological importance that metabolizes numerous xenobiotics to their respective carboxylic acids. Hydrogen peroxide (H2O2) produced as a by-product in this reaction is considered to be involved as a part of the signaling mechanism during differentiation. An observed reduction in the level of H2O2 among AOX1kd cells confirmed production of H2O2 in the reaction catalyzed by AOX1. Taken together, these findings suggest that AOX1 acts as a contributor to the process of myogenesis by influencing the level of H2O2.

Synthesis, characterization, and anti-amoebic activity of N-(pyrimidin-2-yl)benzenesulfonamide derivatives.

A new series of N‐(pyrimidin‐2‐yl)benzenesulfonamide derivatives, 3a–3i and 4a–4i, was synthesized from pyrimidin‐2‐amines, 2a–2i, with the aim to explore their effects on in vitro growth of Entamoeba histolytica. The chemical structures of the compounds were elucidated by elemental analysis, FT‐IR, 1H‐ and 13C‐NMR, and ESI mass‐spectral data. In vitro anti‐amoebic activity was evaluated against HM1 : IMSS strain of Entamoeba histolytica. The IC50 values were calculated by using the double dilution method. The results were compared with the IC50 value of the standard drug ‘metronidazole’. The selected compounds were tested for their cytotoxic activities by cell‐viability assay using H9C2 cardiac myoblasts cell line, and the results indicated that all the compounds displayed remarkable >80% viabilities to a concentration of 100 μg/ml.

Transthyretin: A Transporter Protein Essential for Proliferation of Myoblast in the Myogenic Program

Irregularities in the cellular uptake of thyroid hormones significantly affect muscle development and regeneration. Herein, we report indispensable role of transthyretin (TTR) in maintaining cellular thyroxine level. TTR was found to enhance recruitment of muscle satellite cells to the site of injury, thereby regulating muscle regeneration. Fluorescence-activated cell sorting (FACS) and immunofluorescence analysis of TTRwt (TTR wild type) and TTRkd (TTR knock-down) cells revealed that TTR controlled cell cycle progression by affecting the expression of Cyclin A2. Deiodinase 2 (D2) mediated increases in triiodothyronine levels were found to regulate the expression of myogenic marker, myogenin (MYOG). Moreover, use of a coumarin derivative (CD) revealed a significant reduction in cellular thyroxine, thereby indicating that TTR play a role in the transport of thyroxine. Taken together, these findings suggest that TTR mediated transport of thyroxine represents a survival mechanism necessary for the myogenic program. The results of this study will be highly useful to the strategic development of novel therapeutics to combat muscular dystrophies.

Expression of Transthyretin during bovine myogenic satellite cell differentiation.

Adult myogenesis responsible for the maintenance and repair of muscle tissue is mainly under the control of myogenic regulatory factors (MRFs) and a few other genes. Transthyretin gene (TTR), codes for a carrier protein for thyroxin (T4) and retinol binding protein bound with retinol in blood plasma, plays a critical role during the early stages of myogenesis. Herein, we investigated the relationship of TTR with other muscle-specific genes and report their expression in muscle satellite cells (MSCs), and increased messenger RNA (mRNA) and protein expression of TTR during MSCs differentiation. Silencing of TTR resulted in decreased myotube formation and decreased expression of myosin light chain (MYL2), myosin heavy chain 3 (MYH3), matrix gla protein (MGP), and voltage-dependent L type calcium channel (Cav1.1) genes. Increased mRNA expression observed in TTR and other myogenic genes with the addition of T4 decreased significantly following TTR knockdown, indicating the critical role of TTR in T4 transportation. Similarly, decreased expression of MGP and Cav1.1 following TTR knockdown signifies the dual role of TTR in controlling muscle myogenesis via regulation of T4 and calcium channel. Our computational and experimental evidences indicate that TTR has a relationship with MRFs and may act on calcium channel and related genes.