肌肉特异性微小RNA和成肌调节因子在C2C12细胞成肌分化 过程中的表达

背景：骨骼肌细胞的增殖与分化是由多因素参与的受严格调控的复杂生物学过程,其中,微小RNA和成肌调节因子作为调控基因表达的重要分子在成肌分化过程中起着关键的调控作用。 目的：探讨微小RNA-1,133,206在C2C12细胞成肌分化过程中的表达变化及成肌调节因子的调控作用。 方法：用含体积分数2%马血清的高糖DMEM培养基诱导C2C12细胞体外成肌分化,分别诱导分化后0,1,2,3,4,6 d时提取细胞总RNA,用于real time RT-PCR检测。 结果与结论：倒置显微镜观察和免疫荧光染色显示诱导C2C12细胞分化后3 d开始有肌管和肌球蛋白重链阳性细胞形成,并随诱导时间延长而增多。Real time RT-PCR检测显示,C2C12细胞成肌分化过程中,微小RNA-1,133,206与成肌调节因子MyoD和myogenin mRNA的表达水平均先上升后又恢复至接近分化前水平,而pax7 mRNA的表达则无明显变化。提示肌肉特异性微小RNA及成肌调节因子可能对C2C12细胞的成肌分化发挥一定的调控作用。     

骨形态发生蛋白2诱导成骨分化与长链非编码RNA AK007000的影响

背景：长链非编码RNA调控一系列生理过程,被认为在发育、分化和代谢的基因调控中发挥重要的作用。MC3T3-E1、C2C12和C3H10T1/2细胞可向骨细胞、肌细胞等多个方向分化,用于肌肉骨骼等运动系统相关疾病的研究。 目的：观察长链非编码RNA在骨形态发生蛋白2诱导成骨分化中的作用。 方法：对MC3T3-E1、C2C12和C3H10T1/2细胞在骨形态发生蛋白2诱导下,成骨分化过程中的长链非编码RNA表达变化进行芯片分析,找到在3株细胞中同时变化的长链非编码RNA,siRNA干扰方法观察长链非编码RNA对骨形态发生蛋白2诱导成骨分化的影响,采用Real-Time PCR与碱性磷酸酶染色检测成骨相关指标。 结果与结论：骨形态发生蛋白2诱导MC3T3-E1、C2C12和C3H10T1/2成骨分化过程中,相应成骨指标增高,成肌指标肌细胞生成素降低。筛选出骨形态发生蛋白2诱导成骨分化过程中出发挥作用的长链非编码RNA AK007000。AK007000被干扰后成骨分化指标碱性磷酸酶、骨钙素、RUNX2、SP7表达下降,肌细胞生成素表达上升。因此,AK007000可能具有促进成骨抑制成肌作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

组胺H3受体降低电激发收缩的小鼠成肌细胞胞浆中钙离子浓度

目的:探讨组胺H3受体(H3R)在小鼠成肌细胞C2C12成肌分化过程及分化后的横纹肌细胞中的表达和可能发挥的作用。方法:诱导C2C12细胞成肌分化,测量H3R和分化晚期标志物肌球蛋白重链mRNA和蛋白的表达;分化过程中加入H3R拮抗剂ciproxifan,测量分化早期标志物desmin、中期标志物myogenin和肌球蛋白重链mRNA的表达。Fluo-4结合剂标记分化后的横纹肌胞内钙离子,测量双极交流电200 m A刺激下,H3R激动剂甲基组胺(RMe HA)对胞浆中钙离子浓度的影响。结果:H3R和肌球蛋白重链在成肌分化过程中表达量逐渐增加。Ciproxifan在成肌分化过程中对3种分化标志物mRNA的表达与对照组相比无差异(P0.05)。RMe HA在浓度10 nmol/L~100μmol/L刺激细胞5~20 min,可呈钟形降低因交流电引起的肌浆钙离子浓度的升高(P0.05),其中RMe HA 100 nmol/L在10 min和20 min对电刺激细胞中Ca2+的抑制百分率最高。相同浓度的RMe HA在20 min和10 min时对Ca2+的抑制率比其在5 min时高(P0.05)。结论:H3R可能在成肌分化过程中的作用不大,而在分化成熟细胞中可以降低电刺激引起的胞浆钙离子浓度的升高。     

Satellite cells from dystrophic (mdx) mice display accelerated differentiation in primary cultures and in isolated myofibers.

In the dystrophic (mdx) mouse, skeletal muscle undergoes cycles of degeneration and regeneration, and myogenic progenitors (satellite cells) show ongoing proliferation and differentiation at a time when counterpart cells in normal healthy muscle enter quiescence. However, it remains unclear whether this enhanced satellite cell activity is triggered solely by the muscle environment or is also governed by factors inherent in satellite cells. To obtain a better picture of myogenesis in dystrophic muscle, a direct cell-by-cell analysis was performed to compare satellite cell dynamics from mdx and normal (C57Bl/10) mice in two cell culture models. In one model, the kinetics of satellite cell differentiation was quantified in primary cell cultures from diaphragm and limb muscles by immunodetection of MyoD, myogenin, and MEF2. In mdx cell cultures, myogenin protein was expressed earlier than normal and was followed more rapidly by dual myogenin/MEF2A expression and myotube formation. In the second model, the dynamics of satellite cell myogenesis were investigated in cultured myofibers isolated from flexor digitorum brevis (FDB) muscle, which retain satellite cells in the native position. Consistent with primary cultures, satellite cells in mdx myofibers displayed earlier myogenin expression, as well as an enhanced number of myogenin-expressing satellite cells per myofiber compared to normal. The addition of fibroblast growth factor 2 (FGF2) led to an increase in the number of satellite cells expressing myogenin in normal and mdx myofibers. However, the extent of the FGF effect was more robust in mdx myofibers. Notably, many myonuclei in mdx myofibers were centralized, evidence of segmental regeneration; all central nuclei and many peripheral nuclei in mdx myofibers were positive for MEF2A. Results indicated that myogenic cells in dystrophic muscle display accelerated differentiation. Furthermore, the study demonstrated that FDB myofibers are an excellent model of the in vivo state of muscle, as they accurately represented the dystrophic phenotype.     

Decreased expression of mouse Rbm3, a cold-shock protein, in Sertoli cells of cryptorchid testis

Physiological scrotal hypothermia is necessary for normal spermatogenesis and fertility in mammals. Human RNA binding motif protein 3 (RBM3) is structurally highly similar to the cold-inducible RNA-binding protein (Cirp), and both mRNAs are induced in human cells at the scrotal temperature (32 degrees C). We report here the cloning of mouse Rbm3 cDNA, which encoded an 18-kd protein with 94% identity in amino acid sequence to that of human RBM3. In the testis of adult mice, Rbm3 mRNA and protein were detected in Sertoli cells, but not germ cells, of seminiferous tubules at all stages. The expression was not observed in Sertoli cells of fetuses, but was observed in newborn and older mice. In the TAMA26 mouse Sertoli cell line, the Rbm3 expression level was increased or decreased within 12 hours after temperature shift from 37 degrees C to 32 degrees C or 39 degrees C, respectively. In contrast to Cirp, the cold-induced growth suppression of TAMA26 cells was not affected by suppression of the Rbm3 expression. When mouse testis was exposed to heat stress by experimental cryptorchidism, the level of Rbm3 was decreased in Sertoli cells. Rbm3 may play important roles distinct from those played by Cirp in spermatogenesis and cryptorchidism by regulating the gene expression in Sertoli cells.     

Id2从核迁移到细胞质后通过调节凋亡诱导因子表达促进骨骼肌细胞分化

P>Objective To explore the functional role of Id2 in skeletal muscle regeneration. Methods Id2 expression vectors were transferred into C2C12 cells. The transferred and un-transferred C2C12 skeletal muscle cells were exposed to 50μmol/L H2O2 and 2% horse serum for 12 hours without fetal bovine serum(FBS). Expression of Id2 gene in transferred and untransferred C2C12 cells was observed by RT-PCR. Expression of various myogenesis related proteins in the transferred and untransferred C2C12 cells were observed by Western blotting. Expression of Id2 and AIF proteins in the normal, fiber-damaged and denervated skeletal muscles were observed by immunofluorescence.Results Compared with un-transferred cells, the Id2 transferred cells exhibited higher differentiation. Immunofluorescence staining revealed that 50μmol/L H2O2 treatment increased the expression of nucleic Id2.Under the oxidative stress, Id2 repressed both MyoD repressors and myogenin activator. Two percents of the horse serum, which usually was used to induce myoblasts differentiation, caused most of Id2 proteins translocation from nucleus to cytoplasm. Translocation of Id2 protein from nucleus to cytoplasm inhibited the ROS-induced expression of mitochondrial apoptosis inducing factor(AIF). Immunofluorescence analysis implied that the denervated skeletal muscle showed more increased Id2 and AIF proteins in the nucleus.Conclusion Id2 translocation from nucleus to cytoplasm can accelerate differentiation of skeletal muscle cells. The functional role of Id2 during the skeletal muscle regene     

Myogenin resides in the nucleus and acquires high affinity for a conserved enhancer element on heterodimerization

Myogenin is a member of a family of muscle-specific factors that can activate the muscle differentiation program in nonmyogenin cells. Using antibodies directed against unique domains of myogenin, we show in the present study that myogenin resides in the nucleus of differentiated muscle cells. Myogenin translated in vitro does not exhibit detectable DNA binding activity; however, when dimerized with the ubiquitous enhancer-binding factor E12, it acquires high affinity for an element in the core of the muscle creatine kinase (MCK) enhancer that is conserved among many muscle-specific genes. Antibody disruption experiments show that myogenin, synthesized during differentiation of the BC3H1 and C2 muscle cell lines, is part of a complex that binds to the same site in the MCK enhancer as myogenin-E12 translated in vitro. Mutagenesis of the myogenin-E12-binding site in the MCK enhancer abolishes binding of the heterodimer and prevents trans-activation of the enhancer by myogenin. The properties of myogenin suggest that its functions as a sequence-specific DNA-binding factor that interacts directly with muscle-specific genes during myogenesis. The dependence of myogenin on E12 for high-affinity DNA binding activity also suggests that the susceptibility of various cell types to the actions of myogenin may be influenced by the cellular factors with which it may interact.     

Glutathione depletion impairs myogenic differentiation of murine skeletal muscle C2C12 cells through sustained NF-kappaB activation

Skeletal muscle differentation is a complex process regulated at multiple levels. This study addressed the effect of glutathione (GSH) depletion on the transition of murine skeletal muscle C2C12 myoblasts into myocytes induced by growth factor inactivation. Cellular GSH levels increased within 24 hours on myogenic stimulation of myoblasts due to enhanced GSH synthetic rate accounted for by stimulated glutamate-L-cysteine ligase (also known as gamma-glutamylcysteine synthetase) activity. In contrast, the synthesis rate of GSH using gamma-glutamylcysteine and glutamate as precursors, which reflects the activity of the GSH synthetase, did not change during differentiation. The stimulation of GSH stores preceded the myogenic differentiation of C2C12 myoblasts monitored by expression of muscle-specific genes, creatine kinase (CK), myosin heavy chain (MyHC), and MyoD. The pattern of DNA binding activity of NF-kappaB and AP-1 in differentiating cells was similar both displaying an activation peak at 24 hours after myogenic stimulation. Depletion of cellular GSH levels 24 hours after stimulation of differentiation abrogated myogenesis as reflected by lower CK activity, MyHC levels, MyoD expression, and myotubes formation, effects that were reversible on GSH replenishment by GSH ethyl ester (GHSEE). Moreover, GSH depletion led to sustained activation of NF-kappaB, while GSHEE prevented it. Furthermore, inhibition of NF-kappaB activation restored myogenesis despite GSH depletion. Thus, GSH contributes to the formation of myotubes from satellite myoblasts by ensuring inactivation of NF-kappaB, and hence maintaining optimal GSH levels may be beneficial in restoring muscle mass in chronic inflammatory disorders.     

Immunohistochemical detection of myogenin and p21 in methylcholanthrene-induced mouse rhabdomyosarcomas

3-Methylcholanthrene (MC)-induced 10 embryonal (ERSs) and 24 pleomorphic rhabdomyosarcomas (PRSs) of the dermis in mouse were examined immunohistochemically for myogenin, p21 and proliferating cell nuclear antigen (PCNA) nuclear reactivity and myosin reactivity. ERSs had higher expression of myogenin and p21 compared with that of myosin. PRSs were divided into two groups having high (moderate or marked reactivity; HLM) and low (mild reactivity; LLM) levels of myosin expression. Expression of p21 was higher in HLM-PRSs than in LLM-PRSs. Statistically significant association was observed between myosin and p21 expression in PRSs, but not between myosin and myogenin expression. Myogenin and p21 reactivity were observed in myoblast-like cells, but rarely in multinucleated cells. In ERSs, small undifferentiated myogenic precursor cells were also positive for p21. No difference of PCNA reactivity was observed between HLM-PRSs and LLM-PRSs, although its reactivity was higher in PRSs than in ERSs. The results suggest that myogenin is related to myoblast-like cell differentiation in PRSs and that p21 plays essential roles in myotube formation and myosin expression. In ERSs, p21 may be involved in inhibition of myogenic precursor cell proliferation and differentiation.     

Synergistic regulation of vertebrate muscle development by Dach2, Eya2, and Six1, homologs of genes required for Drosophila eye formation

We have identified a novel vertebrate homolog of the Drosophila gene dachshund, Dachshund2 (Dach2). Dach2 is expressed in the developing somite prior to any myogenic genes with an expression profile similar to Pax3, a gene previously shown to induce muscle differentiation. Pax3 and Dach2 participate in a positive regulatory feedback loop, analogous to a feedback loop that exists in Drosophila between the Pax gene eyeless (a Pax6 homolog) and the Drosophila dachshund gene. Although Dach2 alone is unable to induce myogenesis, Dach2 can synergize with Eya2 (a vertebrate homolog of the Drosophila gene eyes absent) to regulate myogenic differentiation. Moreover, Eya2 can also synergize with Six1 (a vertebrate homolog of the Drosophila gene sine oculis) to regulate myogenesis. This synergistic regulation of muscle development by Dach2 with Eya2 and Eya2 with Six1 parallels the synergistic regulation of Drosophila eye formation by dachshund with eyes absent and eyes absent with sine oculis. This synergistic regulation is explained by direct physical interactions between Dach2 and Eya2, and Eya2 and Six1 proteins, analogous to interactions observed between the Drosophila proteins. This study reveals a new layer of regulation in the process of myogenic specification in the somites. Moreover, we show that the Pax, Dach, Eya, and Six genetic network has been conserved across species. However, this genetic network has been used in a novel developmental context, myogenesis rather than eye development, and has been expanded to include gene family members that are not directly homologous, for example Pax3 instead of Pax6.     

Identification of novel genes expressed during rhabdomyosarcoma differentiation using cDNA microarrays

Rhabdomyosarcomas (RMS) are highly aggressive tumors that are thought to arise as a consequence of the regulatory disruption of the growth and differentiation of skeletal muscle progenitor cells. Normal myogenesis is characterized by the expression of the myogenic regulatory factor gene family but, despite their expression in RMS, these tumor cells fail to complete the latter stages of myogenesis. The RMS cell line RD-A was treated with 12-O-tetradecanoylphorbol-13-acetate to induce differentiation and cultured for 10 days. RNA was extracted on days 1, 3, 6, 8 and 10. A human skeletal muscle cDNA microarray was developed and used to analyze the global gene expression of RMS tumors over the time-course of differentiation. As a comparison, the genes identified were subsequently examined during the differentiated primary human skeletal muscle cultures. Prothymosin alpha (PTMA), and translocase of inner mitochondrial membrane 10 (Tim10), two genes not previously implicated in RMS, showed reduced expression during differentiation. Marked differences in the expression of PTMA and Tim10 were observed during the differentiation of human primary skeletal muscle cells. These results identify several new genes with potential roles in the myogenic arrest present in rhabdomyosarcoma. PTMA expression in RMS biopsy samples might prove to be an effective diagnostic marker for this disease.     

Roles of lncRNAs and circRNAs in regulating skeletal muscle development

The multistep biological process of myogenesis is regulated by a variety of myoblast regulators, such as myogenic differentiation antigen, myogenin, myogenic regulatory factor, myocyte enhancer factor2A‐D and myosin heavy chain. Proliferation and differentiation during skeletal muscle myogenesis contribute to the physiological function of muscles. Certain non‐coding RNAs, including long non‐coding RNAs (lncRNAs) and circular RNAs (circRNAs), are involved in the regulation of muscle development, and the aberrant expressions of lncRNAs and circRNAs are associated with muscular diseases. In this review, we summarize the recent advances concerning the roles of lncRNAs and circRNAs in regulating the developmental aspects of myogenesis. These findings have remarkably broadened our understanding of the gene regulation mechanisms governing muscle proliferation and differentiation, which makes it more feasible to design novel preventive, diagnostic and therapeutic strategies for muscle disorders.