Selection of suitable reference genes for real-time PCR studies of Atlantic halibut development

Gene expression studies are fundamental to understand the molecular basis of severe malformations in fish development, particularly under aquaculture conditions. Real-time PCR (qPCR) is the most accurate method of quantifying gene expression, provided that suitable endogenous controls are used to normalize the data. To date, no reference genes have been validated for developmental gene expression studies in Atlantic halibut (Hippoglossus hippoglossus). We have determined the expression profiles of 6 candidate reference genes (Actb, Eef2, Fau, Gapdh, Tubb2 and 18S rRNA) in 6 embryonic and 5 larval stages of Atlantic halibut development. There were significant changes in expression levels throughout development, which stress the importance and complexity of finding appropriate reference genes. The three software applications (BestKeeper, geNorm and NormFinder) used to evaluate the stability of potential reference genes produced comparable results. Tubb2 and Actb were the most stable genes across the different developmental stages, whereas 18S rRNA and Gapdh were the most variable genes and thus inappropriate to use as reference genes. According to geNorm and NormFinder, the best two-gene normalization factors corresponded to the geometric average of Tubb2/Actb and Tbb2/Fau, respectively. We believe that either of these normalization factors can be used for future developmental gene expression studies in Atlantic halibut.     

The role of histone chaperones in osteoblastic differentiation of C2C12 myoblasts

Cellular differentiation is a process in which the cells gain a more specialized shape, metabolism, and function. These cellular changes are accompanied by dynamic changes in gene expression programs. In most cases, DNA methylation, histone modification, and variant histones drive the epigenetic transition that reprograms the gene expression. Histone chaperones, HIRA and Asf1a, have a role for cellular differentiation by deposition of one of variant histones, H3.3, during myogenesis of murine C2C12 cells. In this study, we accessed the roles of histone chaperones and histone H3.3 in osteoblastic conversion of C2C12 myoblasts and compared their roles with those for myogenic differentiation. The unbiased analysis of the expression pattern of histone chaperones and variant histones proposed their uncommon contribution to each pathway. HIRA and Asf1a decreased to ～50% and further diminished during differentiation into osteoblasts, while they were maintained during differentiation into myotubes. HIRA, Asf1a, and H3.3 were indispensable for expression of cell type-specific genes during conversion into osteoblasts or myotubes. RNA interference analysis indicated that histone chaperones and H3.3 were required for early steps of osteoblastic differentiation. Our results suggest that histone chaperones and variant histones might be differentially required for the distinct phases of differentiation pathway.     

Glucocorticoid inhibition of C2C12 proliferation rate and differentiation capacity in relation to mRNA levels of the MRF gene family

The muscle regulatory factors (MRF) gene family regulate muscle fibre development. Several hormones and drugs also affect muscle development. Glucocorticoids are the only drugs reported to have a beneficial effect on muscle degenerative disorders. We investigated the glucocorticoid-related effects on C2C12 myoblast proliferation rate, morphological differentiation, and subsequent mRNA expression patterns of the MRF genes. C2C12 cells were incubated with the glucocorticoids dexamethasone or alpha-methyl-prednisolone. Both glucocorticoids showed comparable effects. Glucocorticoid treatment of C2C12 cells during the proliferative phase reduced the proliferation rate of the cells dose dependently, especially during the third and fourth day of culture, increased MyoD1, myf-5, and MRF4 mRNA levels, and reduced myogenin mRNA level, compared to untreated control cells. Thus, the mRNA level of proliferation-specific MyoD1 and myf-5 expression does not seem to associate with C2C12 myoblast proliferation rate. Glucocorticoid treatment of C2C12 cells during differentiation reduced the differentiation capacity dose dependently, which is accompanied by a dose dependent reduction of myogenin mRNA level, and increased MyoD1, myf-5, and MRF4 mRNA levels compared to untreated control cells. Therefore, we conclude that glucocorticoid treatment reduces differentiation of C2C12 myoblasts probably through reduction of differentiation-specific myogenin mRNA level, while inducing higher mRNA levels of proliferation-associated MRF genes.     

Pax7基因在C 2C12细胞内的表达及诱导失活(简报)

配对框(Paired box)首先是在果蝇的分节基因中发现的一段DNA保守序列,编码能与DNA特异结合的一个蛋白质结构域。这些序列在进化中有一定的保守性,在许多种生物基因组内存在,其中包括小鼠和人。至今为止,在小鼠中分离到九个含配对框的Pax基因,按基因发现时序,分别定名为Pax 1至Pax 9。Pax 7是其中的一个成员,它不但含有配对框,还含有八肽结构(Octapeptide)和     

Identification of novel GH-regulated genes in C2C12 cells

Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16?h growing in serum-free medium, C2C12 myotubes were stimulated during 6?h with 500?ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function.     

Notch Pathway Activation Contributes to Inhibition of C2C12 Myoblast Differentiation by Ethanol

The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI) abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.     

Nanoparticle-mediated intracellular lipid accumulation during C2C12 cell differentiation

In this report, we sought to elucidate whether multiwall carbon nanotubes are involved in the modulation of the proliferation and differentiation of the skeletal muscle cell line C2C12. Skeletal muscle is a major mass peripheral tissue that accounts for 40% of total body weight and 50% of energy consumption. We focused on the differentiation pathway of myoblasts after exposure to a vapor-grown carbon fiber, HTT2800, which is one of the most highly purified carbon nanotubes. This treatment leads in parallel to the expression of a typical adipose differentiation program. We found that HTT2800 stimulated intracellular lipid accumulation in C2C12 cells. We have also shown by quantified PCR analysis that the expression of adipose-related genes was markedly upregulated during HTT2800 exposure. Taken together, these results suggest that HTT2800 specifically converts the differentiation pathway of C2C12 myoblasts to that of adipoblast-like cells.     

PI3K/AKT信号通路调控Myogenin和MCK基因的表达

骨骼肌分化过程受多个信号通路调控, PI3K/AKT信号通路是其中最重要的信号转导通路之一。PI3K/AKT信号通路可以调控骨骼肌分化, 但在染色质水平上的调控机制还不是很清楚。文章以小鼠成肌细胞(C2C12)为研究材料, 采用免疫印迹、染色质免疫共沉淀(Chromatin immunoprecipitation, ChIP)、定量PCR (Q-PCR)的方法研究PI3K/AKT信号通路调控Myogenin和MCK基因的表达。研究发现, C2C12细胞分化过程中添加PI3K/AKT信号通路激活剂处理24 h, Myogenin和MCK蛋白表达水平显著升高, 组蛋白H3K27me3去甲基化酶UTX的表达也升高, H3K27me3在Myogenin基因启动子区和MCK基因启动子及增强子区的富集与对照组相比显著降低。用PI3K/AKT信号通路抑制剂处理, 结果相反。因此, PI3K/AKT信号通路可能通过调控组蛋白去甲基化酶UTX的表达活性改变靶基因的H3K27me3的富集进而调控骨骼肌分化。     

siRNA沉默Myh3基因抑制C2C12细胞糖酵解

肌球蛋白重链3(myosin heavy chain 3,Myh3)基因为肌肉细胞分化的标志基因,调节肌肉细胞能量的利用,但其是否会影响肌肉细胞不同状态下的糖酵解过程尚鲜有报道。本文以成肌和成脂分化不同阶段的小鼠C2C12细胞为模型,利用qRT-PCR方法研究Myh3与糖酵解相关基因Pkm(M-type pyruvate kinse)、Prkag3(protein kinase adenosine monophosphate-activated γ3-subunit)和Gsk3β(glycogen synthase kinase-3β)的表达模式。发现在C2C12细胞成肌分化过程中,Myh3与糖酵解基因Prkag3和Pkm的相对表达趋势基本一致,都呈现相对表达水平先上升,分化第2 d达到峰值,之后下降的趋势;糖原合酶抑制基因Gsk3β的表达趋势相对平稳。而在C2C12细胞成脂分化过程中,Myh3依然与糖酵解基因Prkag3和Pkm的相对表达趋势基本一致,相对表达量逐渐上升,在分化第8 d达到最高值;糖原合酶抑制基因Gsk3β的表达保持稳定状态。在C2C12细胞成肌分化状态下,qRT-PCR和Western 印迹检测干扰Myh3对细胞糖酵解相关基因Pkm、Prkag3和Gsk3β mRNA和蛋白质表达的影响。结果显示,干扰Myh3后,糖酵解基因Pkm和Prkag3的mRNA表达量极显著降低(P<0.01),糖原合酶抑制基因Gsk3β的mRNA表达无明显变化(P>0.05);Myh3干扰组中Myh3和Pkm的蛋白质水平显著低于空白组和NC组细胞。在C2C12细胞成脂分化状态下,干扰Myh3,糖原合酶抑制基因Gsk3β和糖酵解基因Prkag3的mRNA表达量极显著升高(P<0.01),糖酵解基因Pkm的mRNA表达下降;Myh3干扰组中Myh3和Pkm的蛋白质水平也低于空白组和NC组细胞。综合以上研究,C2C12细胞成肌和成脂状态下糖酵解水平存在明显差异,Myh3与酵解基因的表达模式相似,进一步研究发现,干扰Myh3可以抑制C2C12细胞成肌状态下的糖酵解,不影响糖原合成。与成肌状态不同,在C2C12细胞成脂状态下干扰Myh3,抑制了糖原合成和糖酵解。     

Modulation of proliferation and differentiation of C2C12 skeletal muscle cells by fatty acids

AimsThis study was performed to elucidate whether mitogen-activated protein kinases (MAPKs) are involved in the modulation of the proliferation and differentiation of skeletal muscle cells by fatty acids.Main methodsC2C12 myoblasts were cultured in differentiation medium containing 2% horse serum for 3 days, and treated with each fatty acid. Phosphorylation levels of MAPKs were examined by immunoblot analysis.Key findingsThe mono-unsaturated fatty acids (MUFAs), oleic acid (OA) and n?6 polyunsaturated fatty acids (n?6 PUFAs), linoleic acid (LA), γ-linoleic acid (GLA), and arachidonic acid (AA) increased the proliferation of C2C12 cells. On the other hand, n?3 polyunsaturated fatty acids (n?3 PUFAs) and saturated fatty acids (SFs) did not affect the proliferation of C2C12 cells. In addition, the treatment of cis-9, trans-11 conjugated linoleic acid (c9,t11 CLA) showed an increased cell proliferation. However, trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA) significantly inhibited cell proliferation. Treatment of C2C12 cells with LA, OA, and c9,t11 CLA increased phosphorylation levels of ERK1/2 and JNK during proliferation. During cell differentiation, OA, LA, and c9,t11 CLA stimulated differentiation of C2C12 cells, whereas t10,c12 CLA inhibited differentiation. We also found that OA, LA, and c9, t11 CLA increased phosphorylation level of ERK1/2, but not JNK during differentiation.SignificanceThese results suggest that fatty acids are able to modulate the proliferation and differentiation of skeletal muscle and MAPKs may be involved in the modulation of the proliferation and differentiation of skeletal muscle cells by fatty acids.     

Ryanodine receptor protein is expressed during differentiation in the muscle cell lines BC3H1 and C2C12

BC3H1 and C2C12, murine cell lines, were assessed as model systems for the expression of ryanodine receptor protein during myogenesis. The ryanodine receptor is a calcium release channel of the sarcoplasmic reticulum and a component of the triad junction, a structure which is essential to excitation-contraction coupling in mature striated muscle. BC3H1 and C2C12 cells do not express the ryanodine receptor at detectable levels in a proliferative, nondifferentiated state. The ryanodine receptor protein is expressed during differentiation in BC3H1 and C2C12 cells, becoming detectable within 24 hr of the onset of differentiation. In both cell lines the ryanodine receptor is assembled in oligomeric form and binds [3H]ryanodine with high affinity. Fusion is not required for expression of the ryanodine receptor in either BC3H1 or nonfusing C2C12 cells. The level of expression of the ryanodine receptor protein is modulated by incubation with the growth factors TGF-beta and bFGF in a manner similar to that of other muscle-specific proteins. These initial observations suggest that the BC3H1 and C2C12 cell lines provide a model system for further investigations of the expression and function of the ryanodine receptor during myogenic differentiation.     

Mef2s are required for thick filament formation in nascent muscle fibres

During skeletal muscle differentiation, the actomyosin motor is assembled into myofibrils, multiprotein machines that generate and transmit force to cell ends. How expression of muscle proteins is coordinated to build the myofibril is unknown. Here we show that zebrafish Mef2d and Mef2c proteins are required redundantly for assembly of myosin-containing thick filaments in nascent muscle fibres, but not for the earlier steps of skeletal muscle fibre differentiation, elongation, fusion or thin filament gene expression. mef2d mRNA and protein is present in myoblasts, whereas mef2c expression commences in muscle fibres. Knockdown of both Mef2s with antisense morpholino oligonucleotides or in mutant fish blocks muscle function and prevents sarcomere assembly. Cell transplantation and heat-shock-driven rescue reveal a cell-autonomous requirement for Mef2 within fibres. In nascent fibres, Mef2 drives expression of genes encoding thick, but not thin, filament proteins. Among genes analysed, myosin heavy and light chains and myosin-binding protein C require Mef2 for normal expression, whereas actin, tropomyosin and troponin do not. Our findings show that Mef2 controls skeletal muscle formation after terminal differentiation and define a new maturation step in vertebrate skeletal muscle development at which thick filament gene expression is controlled.     

Effect of miR-143-3p on C2C12 myoblast differentiation

MicroRNAs are a class of 18–22 nucleotide non-coding RNAs that modulate gene expression by associating with the 3′ untranslated regions of mRNAs. A large number of microRNAs are involved in the regulation of myoblast differentiation, many of which remain undiscovered. In this study, we found that miR-143-3p was upregulated during C2C12 myoblast differentiation and over-expression of miR-143-3p significantly inhibited the relative expression levels of MyoD, MyoG, myf5, and MyHC genes, especially in the later stages of differentiation. In addition, miR-143-3p inhibited expression of genes involved in the endogenous Wnt signaling pathway during C2C12 myoblast differentiation, including Wnt5a, LRP5, Axin2, and β-catenin. These results indicate that miR-143-3p represents a new myogenic differentiation-associated microRNA that can inhibit C2C12 myoblast differentiation, especially in the later stages of differentiation.