癌变组织特异高表达抗凋亡蛋白——ARC

带有caspase富集功能域的凋亡抑制因子（ apoptosis repressor with a caspase recuitment domain, ARC ）是新近发现的重要抗凋亡蛋白。在正常组织中,ARC高度特异地表达在终末分化组织如心肌、骨骼肌和大脑,而在非终末分化组织不表达或微量表达。但是,当非终末分化组织发生癌变时表达大量的ARC。ARC过表达可以抑制阿霉素或射线诱导的癌细胞死亡,提示ARC在癌细胞中具有抗趋化或抗放射线损伤的作用。因此,ARC可能成为一种新的肿瘤标记物,也可能是决定肿瘤对各种治疗方法反应性的一个重要因素。     

心脏特异抗凋亡蛋白ARC研究进展

含胱冬肽酶富集功能域的凋亡抑制因子（apoptosis repressor with CARD,ARC）蛋白是20世纪末发现的在心肌、骨骼肌和大脑等终末分化组织中大量表达的抗凋亡蛋白。ARC的主要功能是抗凋亡作用。由于其在心脏中表达的特异性和抗凋亡的多层次性,使ARC在心脏中的作用成为近年来研究的热点。本文主要对其抗凋亡途径以及近年来在心肌病中的研究进展两方面进行综述。     

心脏特异性高度表达的抗凋亡蛋白——ARC

与凋亡(apoptosis)相关的许多心脏疾病如心肌梗死、心肌病及心衰等严重威胁着人类的健康和生命.寻找有效手段防治这些心脏病是当前医学研究的热点.ARC(带有caspase富集功能域的凋亡抑制因子)是新近发现的唯一在心脏大量且特异表达的抗凋亡蛋白,其全称是带有caspase富集功能域的凋亡抑制因子.ARC可被持续性磷酸化并参与阻断凋亡发生途径的多个层面.因此,ARC是一种强大的抗心肌凋亡蛋白.     

甲基转移酶METTL21C影响鸡骨骼肌细胞成肌的发育

本研究旨在探究甲基转移酶METTL21C在家禽骨骼肌发育分化过程中的作用。采用实时荧光定量PCR (quantitative Real-time PCR, qPCR)检测METTL21C基因在鸡不同组织中的表达情况,绘制其组织表达谱;选取3个时间点,检测其在骨骼肌组织中的表达情况。通过酶消化法从鸡骨骼肌组织中分离得到原代细胞;将METTL21C超表达载体转染至原代鸡骨骼肌细胞,通过qPCR和Western blotting检测Pax7、MyoD、Myf5、MyoG等基因的表达水平。结果显示,METTL21C在心肌和骨骼肌组织中的表达量显著高于其他组织,在胚胎期和幼龄期骨骼肌中的表达呈上升趋势;超表达METTL21C后,成肌相关基因Pax7、MyoD、Myf5、MyoG的表达量显著升高。本研究初步发现甲基转移酶METTL21C具有促进家禽骨骼肌发育分化的作用,为骨骼肌发育的分子机理的研究及相关医学研究提供数据支持。     

microRNAs在肌肉中的作用研究进展

microRNAs是一类非蛋白质编码小RNA,通常作用于靶基因mRNA的3′-UTR区引起靶基因的翻译抑制或降解。microRNAs的表达具有组织特异性,骨骼肌和心肌中有特异microRNAs的表达。microRNAs在肌肉的增殖、分化等发育过程中发挥重要的调节作用,并且microRNAs的表达异常与某些肌肉疾病的病理过程有关。现就microRNAs在肌肉中的作用研究进展作一综述。     

lncRNA TCONS_00815878对猪骨骼肌卫星细胞分化的影响

猪骨骼肌发育是一个复杂的生物学过程,其中骨骼肌卫星细胞分化是影响骨骼肌发育的重要环节。近年来发现长链非编码RNA (long non-coding RNA, lncRNA)在骨骼肌卫星细胞分化中具有重要作用。为探究lncRNA TCONS_00815878对猪骨骼肌卫星细胞分化的影响,本研究利用qRT-PCR技术检测出生7 d内大白仔猪6种组织(心脏、脾脏、肺脏、肾脏、背肌和腿肌)及从胚胎期到出生后5个不同时间点(35 d、45 d、55 d胚胎及产后第7 d和第200 d后腿肌肉组织) TCONS_00815878的表达情况;利用反义核苷酸(antisense oligonucleotides, ASO)在猪骨骼肌卫星细胞中敲低TCONS_00815878,检验分化标记基因MyoD、MyoG和MyHC表达情况;通过生物信息学分析预测TCONS_00815878靶基因,并利用DAVID软件在线预测其靶基因的功能与通路。结果表明:TCONS_00815878在猪心肌和腿肌中高表达;仔猪出生后7 d内,TCONS_00815878在猪肌肉组织中表达量不断升高,第7 d达到高峰;在猪骨骼肌卫星细胞增殖和分化过程中,TCONS_00815878在分化期表达量不断上升,且在分化30 h表达量达到峰值;敲低TCONS_00815878后,MyoD、MyoG和MyHC基因表达量降低,其中MyoD表达量显著下降(P0.05)。此外,功能预测结果发现,其靶基因富集到糖酵解和丙酮酸代谢等与骨骼肌卫星细胞分化相关的多个生物学过程。本研究推测,lncRNA TCONS_00815878可能对猪骨骼肌卫星细胞的分化起促进作用。     

冬眠动物骨骼肌生理适应及机制的研究进展

非冬眠动物的骨骼肌在废用条件下会发生明显的萎缩。冬眠动物在历经数月的冬眠期骨骼肌废用后,仍能保持较完整的形态结构与良好的收缩功能,成为天然的抗废用性肌萎缩动物模型。探明冬眠动物骨骼肌对废用的生理适应机制,是生理生态学领域的重要课题之一。本文从形态结构、肌纤维类型和收缩功能等方面综述了冬眠动物对冬眠期骨骼肌废用状态的生理适应,并从蛋白质代谢、生长与分化的调控、代谢类型的调控、氧化应激以及线粒体结构与氧化能力等方面分析了冬眠期骨骼肌生理适应的可能机制。     

心肌再生微环境构建策略

心肌细胞是一种高度分化的终末细胞,自我更新能力差,因而心梗发生后,坏死的心肌细胞不能得到有效的补充,梗死区域很快被纤维组织所取代,严重影响心功能。近年研究发现,利用组织工程手段构建的心肌补片能有效改善心梗区微环境,对心肌的再生能力有着重要的调控作用,能在一定程度上促进心肌再生,缓解心梗状态。该文综述了心肌微环境对心肌再生的调控机制,以及通过心肌补片的手段改善心肌微环境治疗心梗的相关研究,为心肌补片的设计和心梗的治疗提供参考。     

KLHL31促进C2C12细胞的肌原分化

人类KLHL31基因是本实验室已克隆的基因,其蛋白质含有保守的BTB和串连重复的Kelch结构域,已有报道表明其在人类成体骨骼肌和心肌组织中特异表达。RT-PCR分析表明在C2C12细胞肌原分化过程中过表达KLHL31能够提高肌原分化标志基因MyoD与Myogenin的转录水平;荧光报告系统分析发现过表达KLHL31能够增强肌原分化相关基因MCK启动子的活性,表明KLHL31能够促进C2C12细胞的肌原分化。     

干扰素诱导蛋白p204调节心肌分化的机制及应用前景

干扰素诱导蛋白p200家族蛋白包括6种鼠类及4种人类家族成员,具有共同的特征结构,广泛参与调节细胞增殖和分化、衰老和凋亡,在自身免疫反应、抗病毒及抗癌等领域发挥着重要的作用。内源性的p200家族蛋白鼠p204在心肌及骨骼肌表达最高,提示其在肌分化中起着重要作用。本文联系p204的分子结构及调节细胞生长与分化的功能,阐述p204促骨骼肌成肌细胞及胚胎癌细胞分化的机制,及对心肌损伤后心肌再生的应用前景。     

Expression and modification of ARC (apoptosis repressor with a CARD domain) is distinctly regulated by oxidative stress in cancer cells

Apoptosis repressor with a CARD domain (ARC), which has been shown to protect against oxidative stress-induced apoptosis, was initially found to be highly expressed in terminally differentiated tissues like heart and skeletal muscle. Recently, we and others have found that ARC is also expressed at high levels in multiple cancer tissues and cell lines. Here, we compared the regulation of ARC in response to oxidative stress between cancer cells and other types of cells. Similar to cardiomyocyte cell line H9c2 cells, cancer cells with reduced ARC expression were significantly more sensitive to oxidative stress. However, oxidative stress did not down-regulate ARC expression in cancer cells as it did in H9c2 cells. We further found that in H9c2 cells oxidative stress regulates ARC protein expression post-translationally through proteasome-mediated degradation. In cancer cell line HeLa, the majority of ARC exists in phosphorylated state in the absence of oxidative stress, whereas in H9c2 cells only marginal amount of ARC was phosphorylated under similar conditions. Our data suggest that the high level of ARC protein and the constitutive phosphorylation of ARC in cancer cells may play an important role in the protection of cancer cells against oxidative stress.     

Apoptosis repressor with caspase recruitment domain (ARC) inhibits myogenic differentiation

Apoptosis repressor with caspase recruitment domain (ARC), an anti-apoptotic protein, is highly expressed in differentiated heart and skeletal muscle. Apoptosis and differentiation share numerous common pathways; therefore, we examined the impact of ARC on H9c2-myoblast differentiation. We demonstrate that ARC expression levels increase and stabilize upon differentiation. ARC-overexpression in pre-differentiated H9c2-cells suppresses differentiation; indicated by increased myotube formation, nuclear fusion and expression of the differentiation markers myogenin and troponin-T. ARC-overexpression inhibited myoblast differentiation associated caspase-3 activation, suggesting ARC inhibits myogenic differentiation through caspase inhibition. In summary, we show a novel role for ARC in the regulation of muscle differentiation.     

Apoptosis repressor with caspase recruitment domain (ARC) is expressed in cancer cells and localizes to nuclei

Apoptosis repressor with caspase recruitment domain is expressed at high levels in brain and myogenic tissues, consistent with a role to inhibit apoptosis in the terminally differentiated cells. Expression of ARC in cancers is not known. In this study, we reported that ARC was highly expressed in various non-myogenic and non-neurogenic human and rat cancer cell lines. Unexpectedly, ARC was localized almost exclusively to the nuclei of cancer cells, which was unlike the cytoplasmic localization of ARC in non-cancer cells. Furthermore, nuclear ARC in cancer cells did not co-localize with nucleolus protein of 30 kDa, an alternatively spliced ARC isoform. These findings indicate that ARC is distributed differently in cancer cells than non-cancer cells and thus might play a role in neoplastic transformation.     

ARC (apoptosis repressor with caspase recruitment domain) is a novel marker of human colon cancer

The ability of cells to escape apoptosis is critical for carcinogenesis as well as resistance to radiation and chemotherapy. ARC (Apoptosis Repressor with CARD (caspase recruitment domain)) is an unusual inhibitor of apoptosis in that it antagonizes both the extrinsic (death receptor) and intrinsic (mitochondrial/ER) apoptosis pathways. ARC is expressed predominantly in terminally differentiated cells such as cardiac and skeletal myocytes and neurons. Recently, however, the abundance of ARC was noted to be markedly increased in the epithelium of primary human breast cancers compared with benign breast tissue and to confer chemo- and radiation-resistance. Whether the induction of ARC is specific to breast cancer or a more general feature of neoplasia remains unknown. In this study, we assessed the abundance and subcellular localization of ARC in 21 human colon cancer cell lines and in 44 primary human colon adenocarcinomas and adjacent benign colonic tissue. ARC was present at high levels in most colon cancer cell lines and in almost all primary colon cancers compared with corresponding controls. Levels of ARC in the cytoplasm were increased in well, moderately, and poorly differentiated cancers compared with benign tissue, while levels of nuclear ARC were increased only in moderately differentiated tumors. Moreover, epithelial cancers of the ovary and cervix exhibited increased ARC abundance compared with controls. These results demonstrate that ARC is a novel marker of human colon cancer and suggest that it may be a feature of epithelial cancers.     

Apoptosis repressor with caspase recruitment domain is dramatically reduced in cardiac, skeletal, and vascular smooth muscle during hypertension

Apoptosis repressor with caspase recruitment domain (ARC) is a unique anti-apoptotic protein with a distinct tissue distribution. In addition, unlike most anti-apoptotic proteins which act on one pathway, ARC can inhibit apoptosis mediated by both the death-receptor and mitochondrial signaling pathways. In this study, we confirm previous reports showing high levels of ARC protein in rat heart and skeletal muscle, but demonstrate for the first time that ARC is also expressed in rat aorta. Immunoblot analysis on endothelium-denuded aorta as well as immunohistochemical analysis on intact aorta demonstrated that ARC was highly expressed in smooth muscle. Immunoblot analysis also found that ARC protein was severely downregulated in skeletal muscle (−82%; P < 0.001), heart (−80%; P < 0.001), and aorta (−71%; P < 0.001) of spontaneously hypertensive rats (SHR) compared to normotensive Wistar-Kyoto (WKY) rats. Decreased ARC levels were also confirmed in tissues of hypertensive animals by immunohistochemical analysis. Collectively, this data suggests that ARC protein is expressed in vascular smooth muscle and is significantly reduced in several target tissues during hypertension.     

Development of a ponasterone A-inducible gene expression system for application in cultured skeletal muscle cells

The goal of this study was to develop an inducible gene expression system to assess functions of specific proteins in differentiated cultured skeletal muscle. We utilized and modified the ecdysone inducible system because others have used this system to express exogenous genes in vitro and in transgenic animals. A limitation of the commercially-available ecdysone system is its constitutive expression in all tissues. Hence, its application in vivo would result in expression of a cloned gene in undifferentiated and differentiated tissues. To target its expression to muscle, we removed the constitutively-active CMV promoter of pVgRXR and replaced it with a skeletal muscle alpha-actin promoter so that the regulatory features of the system would be expressed in differentiated muscle cells. We transfected our newly designed expression system into L8 muscle myoblasts and established stable cell lines via antibiotic selection. We determined that reporter gene activity was induced by ponasterone A in myotubes, a differentiated muscle phenotype, but not in myoblasts (undifferentiated cells). This proved the validity of the concept of an inducible muscle-specific expression system. We then determined that beta-galactosidase expression was dependent upon the dose of ponasterone A and duration of exposure to inducer. This creates potential to regulate both the level of expression and duration of expression of a cloned gene in differentiated muscle.     

Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice.

Muscle creatine kinase (MCK) is expressed at high levels only in skeletal and cardiac muscle tissues. Previous in vitro transfection studies of skeletal muscle myoblasts and fibroblasts had identified two MCK enhancer elements and one proximal promoter element, each of which exhibited expression only in differentiated skeletal muscle. In this study, we have identified several regions of the mouse MCK gene that are responsible for tissue-specific expression in transgenic mice. A fusion gene containing 3,300 nucleotides of MCK 5' sequence exhibited chloramphenicol acetyltransferase activity levels that were more than 10(4)-fold higher in skeletal muscle than in other, nonmuscle tissues such as kidney, liver, and spleen. Expression in cardiac muscle was also greater than in these nonmuscle tissues by 2 to 3 orders of magnitude. Progressive 5' deletions from nucleotide -3300 resulted in reduced expression of the transgene, and one of these resulted in a preferential decrease in expression in cardiac tissue relative to that in skeletal muscle. Of the two enhancer sequences analyzed, only one directed high-level expression in both skeletal and cardiac muscle. The other enhancer activated expression only in skeletal muscle. These data reveal a complex set of cis-acting sequences that have differential effects on MCK expression in skeletal and cardiac muscle.