增强子RNA研究现状

增强子是真核生物基因表达调控的主要顺式作用元件,能有效促进基因表达。活化的增强子可以转录生成增强子RNA (enhancer RNAs, eRNAs),其合成受到信号系统和信号转录因子的约束。eRNAs与其他转录本(如lncRNAs和mRNAs)相比,其长度更短、稳定性更差、组织特异性更强。此外,eRNAs对增强子与启动子之间的染色质环(looping)的形成和稳定有一定的作用,并能促进靶基因的表达。目前,越来越多的研究发现eRNAs在发育和疾病发生等生物学过程中扮演着重要角色,但是其功能研究一直进展缓慢,调控机制尚不清楚。本文概述了eRNAs的特征、研究方法和功能特性,探讨了eRNAs作为潜在治疗靶标的可能性,以期为eRNAs的后续研究提供参考。     

The NTR module: domains of netrins, secreted frizzled related proteins, and type I procollagen C-proteinase enhancer protein are homologous with tissue inhibitors of metalloproteases.

Using homology search, structure prediction, and structural characterization methods we show that the C-terminal domains of (1) netrins, (2) complement proteins C3, C4, C5, (3) secreted frizzled-related proteins, and (4) type I procollagen C-proteinase enhancer proteins (PCOLCEs) are homologous with the N-terminal domains of (5) tissue inhibitors of metalloproteinases (TIMPs). The proteins harboring this netrin module (NTR module) fulfill diverse biological roles ranging from axon guidance, regulation of Wnt signaling, to the control of the activity of metalloproteases. With the exception of TIMPs, it is not known at present what role the NTR modules play in these processes. In view of the fact that the NTR modules of TIMPs are involved in the inhibition of matrixin-type metalloproteases and that the NTR module of PCOLCEs is involved in the control of the activity of the astacin-type metalloprotease BMP1, it seems possible that interaction with metzincins could be a shared property of NTR modules and could be critical for the biological roles of the host proteins.     

Stabilization of RNA strands in protein synthesis by type I procollagen C-proteinase enhancer protein, a potential RNA-binding protein, in hepatic stellate cells.

Type I procollagen C-proteinase enhancer (PCPE) exists in hepatic stellate cells (HSCs) which can produce collagen. The deduced amino acid sequence of PCPE contains motifs specific for RNA-binding proteins. The effect of PCPE on the syntheses of collagen and noncollagenous protein was studied using an HSC clone derived from cirrhotic rat liver. When the cells were cultured in the presence of an antisense oligonucleotide (AS) against PCPE mRNA, the synthesis of noncollagenous protein as well as collagen was reduced compared to the cells cultured with addition of a nonsense oligonucleotide (NS). The extent of the reduction was similar in both syntheses. The total RNA content of the AS-treated cells and NS-treated cells did not differ. In the presence of actinomycin D, however, such total RNA content was decreased more rapidly in the AS-treated cells than in the NS-treated cells. PCPE may be involved in stabilization of RNA strands in noncollagenous protein synthesis as well as collagen synthesis.     

Lef-1基因在棕色和白色羊驼皮肤差异表达

本实验旨在研究Lef-1在不同毛色羊驼皮肤中的表达和定位,探索其与毛色间的关系.采用实时荧光定量PCR、Western blotting以及免疫组织化学方法,研究白色和棕色羊驼皮肤中的mRNA、蛋白表达水平和定位.实时荧光定量PCR结果显示,Lef-1在棕色羊驼皮肤组织相对基因表达量是3.3727±0.1989,在白色羊驼皮肤组织是1.0003±0.0227; Western blotting结果显示,在羊驼皮肤组织总蛋白中存在分子量约44 KD与兔抗Lef-1多克隆抗体发生免疫阳性反应的蛋白条带,棕色羊驼平均蛋白表达量显著高于白色羊驼;免疫组织化学结果显示,在棕色羊驼皮肤组织中多表达在毛球部、表皮也有分布,在白色羊驼皮肤组织中多表达在表皮,在棕色和白色羊驼皮肤组织中外根鞘都有较强表达.结果显示Lef-1在棕色和白色羊驼皮肤的定位和含量存在差异,提示Lef-1可能影响了羊驼被毛颜色的形成.     

Sigma54依赖性转录起始

作为细菌RNA聚合酶(RNAP)的组成型辅助因子,sigma70和sigma54分别参与了原核细胞不同类型基因的转录起始调控。sigma70负责管家基因的自发转录起始;而sigma54负责应激信号相关的基因转录起始。sigma54与RNAP形成复合物后,会通过空间阻滞的方式阻碍DNA进入RNAP中,抑制基因转录起始。当细胞环境变化后,特定应激信号会通过细菌增强子结合蛋白(bEBP)诱发sigma54的构象发生变化,解除sigma54对RNAP的抑制,启动sigma54依赖的基因转录。最近的结构生物学研究揭示了sigma54依赖性转录起始的若干复合物结构,包括全酶、封闭式复合物、2个中间状态复合物及开放式复合物。通过分析这些转录起始复合物的结构,本文阐述了转录起始过程中复合物的结构变化。描述并分析了sigma54和bEBP在转录起始过程中所发挥的功能。本文有助于了解转录起始分子水平的变化,为深入理解sigma54和bEBP促进转录起始的分子机制提供了参考。     

miR-101a靶向EZH2促进山羊骨骼肌卫星细胞的分化

本实验室前期研究发现,miR-101a对山羊骨骼肌卫星细胞(skeletal muscle satellite cells, SMSCs)分化有促进作用,但其具体作用机制并不清楚。本研究利用PicTar、TargetScan和miRanda软件在线预测miR-101a的靶基因,并通过双荧光素酶报告基因进行实验验证;检测了山羊SMSCs分化不同时期miR-101a和靶基因的表达关系,同时分析了超表达和抑制miR-101a对靶基因表达水平的影响。结果证实,zeste增强子同源物2(enhancer of zeste homologue 2, EZH2)基因mRNA的3°UTR具有miR-101a结合位点,是miR-101a的一个靶基因。在SMSCs分化过程中,随着miR-101a表达水平的升高,EZH2的表达在mRNA和蛋白水平均下调。抑制miR-101a后,EZH2的表达极显著升高(P<0.01),但是在超表达miR-101a条件下,EZH2表达变化在mRNA和蛋白水平均不显著(P>0.05)。以上研究结果表明,miR-101a能通过抑制EZH2的表达来促进山羊SMSCs分化,为进一步阐明miR-101a对SMSCs的调控机制提供了理论依据。     

Immediate–Early(IE) gene regulation of cytomegalovirus:IE1-and pp71-mediated viral strategies against cellular defenses

Three crucial hurdles hinder studies on human cytomegalovirus(HCMV): strict species specificity, differences between in vivo and in vitro infection, and the complexity of gene regulation. Ever since the sequencing of the whole genome was first accomplished, functional studies on individual genes have been the mainstream in the CMV field. Gene regulation has therefore been elucidated in a more detailed fashion. However, viral gene regulation is largely controlled by both cellular and viral components. In other words, viral gene expression is determined by the virus–host interaction. Generally, cells respond to viral infection in a defensive pattern; at the same time, viruses try to counteract the cellular defense or else hide in the host(latency). Viruses evolve effective strategies against cellular defense in order to achieve replicative success. Whether or not they are successful, cellular defenses remain in the whole viral replication cycle: entry, immediate–early(IE) gene expression, early gene expression, DNA replication, late gene expression, and viral egress. Many viral strategies against cellular defense, and which occur in the immediate–early time of viral infection, have been documented. In this review, we will summarize the documented biological functions of IE1 and pp71 proteins, especially with regard to how they counteract cellular intrinsic defenses.     

超级增强子在肿瘤研究中的进展

超级增强子是由多个相邻近的普通增强子组成的、驱动调控细胞身份基因表达的一个大簇,该区域富集高密度的转录因子、辅因子及增强子相关表观修饰。超级增强子所驱动的异常转录基因对维持肿瘤细胞特性至关重要。肿瘤细胞通过组装自身超级增强子,显著促进多种癌基因表达,从而增强肿瘤细胞的增殖、侵袭和转移的能力;抑制超级增强子的活性,则显著抑制肿瘤细胞的生长和存活。本文对目前报道的肿瘤细胞中超级增强子的结构特征和功能调控,以及靶向超级增强子药物研发现状进行了总结,旨在为研发新的针对超级增强子为靶点的抗肿瘤药物提供理论基础和借鉴。     

利用生物信息学方法对人类(Homo sapiens)肌肉增强因子2(MEF2)的特性比较及进化分析

肌肉增强因子2(Myocyte enhancer factor 2,MEF2)是MADS(MCM1,agamous,deficiens和serum response factor)家族成员之一,在动物发育过程中起到重要的调节作用。为了进一步了解其调控的复杂性,本文根据NCBI中已有的人类MEF2相关数据,应用ExPASy在线序列分析工具、CBS在线分析服务器软件、Conserved Domain Database(CDD)数据库、SABLE在线分析软件等对人类MEF2蛋白的不同亚型序列进行比较分析,同时,根据相关序列的比对结果构建系统进化树进行分析。结果表明,MEF2在人体内以多种蛋白形式存在,其理化性质存在一定差别,可能的翻译后糖基化修饰多为O型糖基化且均存在较多磷酸化位点。人类各MEF2蛋白具明显MADS结构域,多数具有MEF2结构域和HJURP_C结构域。各MEF2蛋白二级结构均包括了螺旋、折叠和无规则卷曲等多种形式,其三级结构模式相似。系统进化树显示MEF2B蛋白与其他蛋白有着较大的序列差异及较远进化关系,可能较为原始。     

Green fluorescent protein (GFP) expression patterns in the olfactory epithelium of GFP transgenic cloned Jinhua pigs

Domestic pigs possess a well‐developed sense of smell. However, the morphology of the porcine olfactory epithelium (OE) is poorly understood. Recently, several strains of transgenic cloned pigs that are presumed to ubiquitously express green fluorescent protein (GFP) have been created. Thus, the purpose of this study was to elucidate the features of porcine OE using the tissues of GFP transgenic cloned pigs. Based on observations of Hematoxylin and Eosin staining and measurements of thickness, porcine OE tissue portions were classified into three categories (thick, standard, and thin). Cryosections revealed that the prominent GFP signals were expressed in olfactory sensory neurons (OSN), Bowman's glands, and olfactory nerve. A few GFP‐expressing sustentacular cells were seen; however, the intensity of GFP fluorescence was slight. In the thick portion, numerous GFP‐expressing polygonal OSN that did not possess dendrites were found. In the standard portions, GFP‐expressing cells had longitudinal dendrites. A few GFP‐expressing cells were found in the thin portion. In the thick and standard portions, most of the prominent GFP‐expressing cells were positive for olfactory marker protein. Moreover, double immunofluorescence staining with boiled GFP and Sox2 antibody revealed that GFP expression patterns in OSN are synchronized with Sox2 immunoreactive patterns.     

沉默脂肪细胞增强子结合蛋白2通过PI3K/Akt途径抑制肝癌细胞增殖和迁移

脂肪细胞增强子结合蛋白2(AEBP2)作为多梳抑制复合物2(PRC2)的组成蛋白质,参与多种肿瘤细胞的增殖和迁移,然而其在肝癌中的作用尚不清楚。本研究基于UALCAN和Kaplan-Meier Plotter数据库分析发现,AEBP2在肝癌组织中高表达,并且与患者的不良预后呈正相关。实时荧光定量PCR和蛋白质印迹结果证实,AEBP2在肝癌细胞中的表达高于正常肝细胞。在HepG2和Huh-7细胞中转染AEBP2 siRNA,平板克隆、CCK-8、流式细胞术、划痕愈合和Transwell结果显示,沉默AEBP2可以抑制肝癌细胞增殖、迁移和侵袭,并促进细胞凋亡(P＜0.05)。免疫荧光检测和蛋白质印迹结果显示,沉默AEBP2能够抑制肝癌细胞上皮-间质转化(EMT)(P＜0.05)。生物信息学分析结果表明,AEBP2参与调控PI3K/Akt信号通路。蛋白质印迹结果证实,沉默AEBP2能下调PI3K、p-AKT (S473)、mTOR、MMP-2和MMP-9的蛋白质表达水平(P＜0.05)。此外,沉默AEBP2对HepG2细胞迁移和侵袭的影响可被PI3K/Akt通路激动剂胰岛素样生长因子1(IGF-1)部分逆转(P＜0.01)。综上所述,AEBP2可能通过调节PI3K/Akt途径促进肝癌细胞增殖和迁移。本研究为AEBP2在肝癌中的作用提供理论依据。     

LncRNA SNHG17 promotes gastric cancer progression by epigenetically silencing of p15 and p57

Long noncoding RNAs (lncRNA) are attractive biomarkers and therapeutic targets because of their disease- and stage-restricted expression. Small nucleolar RNA host gene 17 (SNHG17) belongs to a large family of noncoding genes hosting small RNAs, with its expression pattern and biological function not clarified in gastric cancer (GC). Thus, we conducted this study to investigate the functional significance and the underlying mechanisms of SNHG17 in GC progression. Our results showed that SNHG17 expression was upregulated in GC tissues and cells, and its high expression was significantly correlated with increased invasion depth, lymphatic metastasis, and advanced TNM stage. The expression of plasma SNHG17 was also found upregulated in patients with GC compared with healthy controls, with a moderate accuracy for diagnosis of GC (area under the receiver operating characteristic curve = 0.748; 95% CI, 0.666–0.830). Gain- and loss-of-function of SNHG17 revealed that SNHG17 promoted GC cell proliferation, cell cycle progression, invasion, and migration and inhibited apoptosis. Mechanistic investigations showed that SNHG17 was associated with polycomb repressive complex 2 and that this association was required for epigenetic repression of cyclin-dependent protein kinase inhibitors, including p15 and p57, thus contributing to the regulation of GC cell cycle and proliferation. Furthermore, rescue experiments indicated that SNHG17 functioned as an oncogene via activating enhancer of zeste homolog 2 in GC cells. Our study provides a new perspective for SNHG17 acting as a noncoding oncogene in GC tumorigenesis, and it may serve as a novel early diagnostic marker and potential target for the treatment of GC.     

Cis-acting elements regulate alternative splicing of exons 6A, 6B and 8 of the α1(XI) collagen gene and contribute to the regional diversification of collagen XI matrices

Consecutive exons 6A, 6B, 7 and 8 that encode the variable region of the amino-terminal domain (NTD) of the col11a1 gene product undergo a complex pattern of alternative splicing that is both tissue-dependent and developmentally regulated. Expression of col11a1 is predominantly associated with cartilage where it plays a critical role in skeletal development. At least five splice-forms (6B-7-8, 6A-7-8, 7-8, 6B-7 and 7) are found in cartilage. Splice-forms containing exon 6B or 8 have distinct distributions in the long bone during development, while in non-cartilage tissues, splice-form 6A-7-8 is typically expressed. In order to study this complex and tissue-specific alternative splicing, a mini-gene that contains mouse genomic sequence from exon 5 to 11, flanking the variable region of α1(XI)-NTD, was constructed. The minigene was transfected into chondrocytic (RCS) and non-chondrocytic (A204) cell lines that endogenously express α1(XI), as well as 293 cells which do not express α1(XI). Alternative splicing in RCS and A204 cells reflected the appropriate cartilage and non-cartilage patterns while 293 cells produced only 6A-7-8. This suggests that 6A-7-8 is the default splicing pathway and that cell or tissue-specific trans-acting factors are required to obtain pattern of the alternative splicing of α1(XI) pre-mRNA observed in chondrocytes. Deletional analysis was used to identify cis-acting regions important for regulating splicing. The presence of the intact exon 7 was required to generate the full complex chondrocytic pattern of splicing. Furthermore, deletional mapping of exon 6B identified sequences required for expression of exon 6B in RCS cells and these may correspond to purine-rich (ESE) and AC-rich (ACE) exonic splicing enhancers.