The Circular RNA Cdr1as Promotes Myocardial Infarction by Mediating the Regulation of miR-7a on Its Target Genes Expression

Objectives

Recent studies have demonstrated the role of Cdr1as (or CiRS-7), one of the well-identified circular RNAs (circRNAs), as a miR-7a/b sponge or inhibitor in brain tissues or islet cells. This study aimed to investigate the presence of Cdr1as/miR-7a pathway in cardiomyocytes, and explore the mechanism underlying the function of miR-7a in protecting against myocardial infarction (MI)-induced apoptosis.

Methods

Mouse MI injury model was established and evaluated by infarct size determination. Real-time PCR was performed to quantify the expression of Cdr1as and miR-7a in cardiomyocytes. Cell apoptosis was determined by caspase-3 activity analysis and flow cytometry assays with Annexin V/PI staining. Transfection of Cdr1as overexpressing plasmid and miR-7a mimic were conducted for gain-of-function studies. Luciferase reporter assay and western blot analysis were performed to verity potential miR-7a targets.

Results

Cdr1as and miR-7a were both upregulated in MI mice with increased cardiac infarct size, or cardiomyocytes under hypoxia treatment. Cdr1as overexpression in MCM cells promoted cell apoptosis, but was then reversed by miR-7a overexpression. The SP1 was identified as a new miR-7a target, in line with previously identified PARP, while miR-7a-induced decrease of cell apoptosis under hypoxia treatment was proven to be inhibited by PARP-SP1 overexpression. Moreover, Cdr1as overexpression in vivo increased cardiac infarct size with upregulated expression of PARP and SP1, while miR-7a overexpression reversed these changes.

Conclusions

Cdr1as also functioned as a powerful miR-7a sponge in myocardial cells, and showed regulation on the protective role of miR-7a in MI injury, involving the function of miR-7a targets, PARP and SP1.     

miR-378促进脂质生成相关靶基因鉴定

旨为验证miR-378在脂肪细胞中的功能,及其脂质相关靶基因的筛选和鉴定.利用miR-378类似物转染3T3-L1细胞,验证miR-378在脂肪细胞中的功能;根据靶标位点的保守性以及促脂功能确定miR-378潜在靶基因;采用microRNA pulldown技术验证miR-378与靶基因的靶标关系;运用双荧光素报告基因...     

miR-15a靶基因的预测及生物信息学分析

目的:对目前研究较为广泛的miR-15a的靶基因进行预测及相关生物信息学分析,以期为miR-15a靶基因的实验验证提供数据支持,并为深入研究miR-15a的调控机制及生物学功能奠定基础和提供理论指导。方法:选择TargetScan5.1与PicTar两种计算方法预测miR-15a的靶基因的交集作为分析的基因集合,分别进行GO注释描述、GO富集分析和生物通路富集分析。结果与结论:预测靶基因集合分别富集在转录调控、蛋白质修饰、细胞周期等生物学过程和蛋白激酶活性等分子功能上(P0.01);经典miR-15a预测靶基因集合显著富集于KEGG通路数据库中的Wnt信号通路、细胞周期和p53信号通路等5个信号转导通路及前列腺癌、慢性髓细胞性白血病、黑素瘤等7个疾病通路中(P0.05)。     

miR-29a靶基因预测及其相关生物信息学分析

目的:通过对miR-29a进行靶基因预测及相关生物信息学分析,为miR-29a靶基因的实验验证提供数据支持,以期为深入研究miR-29a的生物学功能和调控机制提供理论指导。方法:利用PubMed检索miR-29a相关文章,通过miRBase在线工具分析miR-29a序列。应用TargetScan及miRNAda两种计算方法预测miR-29a靶基因并取其交集作为分析的基因集合,分别进行基因本体(gene ontology,GO)中的分子功能和生物学过程以及KEGG(Kyoto Encyclopedia of Genes and Genomes)生物通路富集分析。结果:(1)miR-29a序列在多物种间具有高度保守性。(2)两种方法预测miR-29a靶基因交集共191个。(3)miR-29a靶基因GO分子功能集中于转录因子活性、DNA结合和钙离子结合等(P0.05);miR-29a靶基因GO生物学过程集中于调控转录、细胞粘附、细胞增殖与凋亡等(P0.05);KEGG生物通路主要富集于PI3K-AKT信号通路、JAK-STAT信号通路、T细胞受体信号通路和胰岛素信号通路等信号转导通路,以及肺小细胞癌和子宫内膜癌等疾病通路(P0.05)。结论:miR-29a可能通过参与多个靶基因信号通路的调控,在机体的多种生理病理过程中发挥重要作用,是一个颇有研究价值的生物学靶标。     

A Clinically Orientated Approach for Combining Discordant and Concordant Sib Pairs

The use of extreme discordant sib pairs (EDSP) or extreme concordant sib pairs (ECSP) has recently been proposed to increase power for mapping quantitative traits in humans (RISCH and ZHANG, 1995, 1996). In this paper we propose a test statistic to jointly analyze EDSP and ECSP based on a clinical sampling procedure. This test statistic does not fulfill any optimality criteria. However, this approach is useful for quantitative traits of clinical significance for which EDSP are rare and/or expensive to ascertain. We show how sample size calculations can be adjusted for recombination using single markers, multipoint analysis, incompletely polymorphic markers and varying proportions of ECSP. If the true genetic model is unknown, the combined approach appears to be more robust than sampling based on only EDSP or only ECSP. We discuss how to find the optimal proportion of EDSP and ECSP to be included in an analysis under power considerations.     

miR-171基因家族进化分析及靶基因预测

运用生物信息学方法在miRBase中搜索植物miR-171基因家族的序列,分析miR-171序列的进化特征并预测其靶基因。结果表明,在38种植物中共搜索到219条miR-171序列,大部分miR-171基因都存在于基因间隔区。进化分析表明,miR-171基因家族的进化与物种进化关联不大。采用3个miRNA靶基因预测软件对大豆和玉米miR-171基因的靶基因进行预测,发现了miR-171可能参与生长因子、转录因子、蛋白酶等的调控,作用范围非常广泛。     

miR-335在肿瘤组织中的表达及其预测靶基因的生物信息学分析

目的:分析miR-335在多种肿瘤组织与癌旁组织中的表达,预测其靶基因并进行相关生物信息学分析,为进一步研究miR-335在肿瘤中的调控机制提供理论基础。方法:分析miR-335的保守性及在多个肿瘤组织中的表达;预测miR-335靶基因,并使用DAIVID对miR-335靶基因进行生物信息学分析。结果:miR-335序列高度保守,在肝癌、肺癌、乳腺癌、肝内胆管癌、脂肪肉瘤中表达下调(P<0.05)。预测miR-335靶基因共34个,靶基因集合功能富集于细胞迁移、凋亡、转录调控,以及蛋白质分子连接、细胞骨架组成等生物学过程和分子功能(P<0.05);主要参与了轴突向导和黏着斑信号通路、黑素瘤疾病信号通路及TGF-β信号通路(P<0.05)。结论:miR-335在多种肿瘤中表达异常,且涉及多个生物学过程和信号转导通路,与肿瘤的发生发展密切相关。     

PLOD3基因3′调控区的人类特异突变改变miR-124a对PLOD3的调控

microRNAs(miRNAs)是一类在转录后水平调控基因表达的不编码蛋白质的小RNA(长度20—24个碱基)。其中,miR-124a是一个在哺乳动物中枢神经系统高度表达的miRNA,在神经前体细胞向神经元分化的过程中起着举足轻重的作用。由于miRNAs特异性地识别靶基因的3′端调控区(3′UTR)的靶序列,因此,在人类起源过程中基因3′UTR的单核苷酸序列变异有可能导致miRNA调控的改变。通过靶基因预测和3′UTR区在哺乳动物代表物种间的同源序列比较,我们发现miR-124a的靶基因中有一个基因(PLOD3)3′UTR的靶位点中存在人类特异突变位点。利用体外报告基因系统,发现PLOD3基因3′UTR靶位点中所含的一个人类特异的突变导致miR-124a对PLOD3的调控效率降低。研究表明,miRNAs靶基因3′UTR的序列变异具有功能效应,它有可能是人类中枢神经系统在起源和演化中发挥关键作用的重要遗传机制之一。     

PLOD3基因3’调控区的人类特异突变改变miR-124a对PLOD3的调控

microRNAs(miRNAs)是一类在转录后水平调控基因表达的不编码蛋白质的小RNA(长度20-24个碱基).其中,miR-124a是一个在哺乳动物中枢神经系统高度表达的miRNA,在神经前体细胞向神经元分化的过程中起着举足轻重的作用.由于miRNAs特异性地识别靶基因的3'端调控区(3'UTR)的靶序列,因此,在人类起源过程中基因3'UTR的单核苷酸序列变异有可能导致miRNA调控的改变.通过靶基因预测和3'UTR区在哺乳动物代表物种间的同源序列比较,我们发现miR-124a的靶基冈中有一个基因(PLOD3)3UTR的靶位点中存在人类特异突变位点.利用体外报告基因系统,发现PLOD3基因3'UTR靶位点中所含的一个人类特异的突变导致miR-124a对PLOD3的调控效率降低.研究表明,miRNAs靶基因3'UTR的序列变异具有功能效应,它有可能足人类中枢神经系统在起源和演化中发挥关键作用的重要遗传机制之一.     

Differential Regulation of Actin Polymerization and Structure by Yeast Formin Isoforms

The budding yeast formins, Bnr1 and Bni1, behave very differently with respect to their interactions with muscle actin. However, the mechanisms underlying these differences are unclear, and these formins do not interact with muscle actin in vivo. We use yeast wild type and mutant actins to further assess these differences between Bnr1 and Bni1. Low ionic strength G-buffer does not promote actin polymerization. However, Bnr1, but not Bni1, causes the polymerization of pyrene-labeled Mg-G-actin in G-buffer into single filaments based on fluorometric and EM observations. Polymerization by Bnr1 does not occur with Ca-G-actin. By cosedimentation, maximum filament formation occurs at a Bnr1:actin ratio of 1:2. The interaction of Bnr1 with pyrene-labeled S265C Mg-actin yields a pyrene excimer peak, from the cross-strand interaction of pyrene probes, which only occurs in the context of F-actin. In F-buffer, Bnr1 promotes much faster yeast actin polymerization than Bni1. It also bundles the F-actin in contrast to the low ionic strength situation where only single filaments form. Thus, the differences previously observed with muscle actin are not actin isoform-specific. The binding of both formins to F-actin saturate at an equimolar ratio, but only about 30% of each formin cosediments with F-actin. Finally, addition of Bnr1 but not Bni1 to pyrene-labeled wild type and S265C Mg-F actins enhanced the pyrene- and pyrene-excimer fluorescence, respectively, suggesting Bnr1 also alters F-actin structure. These differences may facilitate the ability of Bnr1 to form the actin cables needed for polarized delivery of nutrients and organelles to the growing yeast bud.Bni1 and Bnr1 are the two formin isoforms expressed in Saccharomyces cerevisiae (1, 2). These proteins, as other isoforms in the formin family, are large multidomain proteins (3, 4). Several regulatory domains, including one for binding the G-protein rho, are located at the N-terminal half of the protein (4–7). FH1, FH2, and Bud6 binding domains are located in the C-terminal half of the protein (8). The formin homology 1 (FH1)² domain contains several sequential poly-l-proline motifs, and it interacts with the profilin/actin complex to recruit actin monomers and regulate the insertion of actin monomers at the barbed end of actin (9–11). The fomin homology domain 2 (FH2) forms a donut-shaped homodimer, which wraps around actin dimers at the barbed end of actin filaments (12, 13). One important function of formin is to facilitate actin polymerization by stabilizing actin dimers or trimers under polymerization conditions and then to processively associate with the barbed end of the elongating filament to control actin filament elongation kinetics (13–18).A major unsolved protein in the study of formins is the elucidation of the individual functions of different isoforms and their regulation. In vivo, these two budding yeast formins have distinct cellular locations and dynamics (1, 2, 19, 20). Bni1 concentrates at the budding site before the daughter cell buds from the mother cell, moves along with the tip of the daughter cell, and then travels back to the neck between daughter and mother cells at the end of segregation. Bnr1 localizes only at the neck of the budding cell in a very short period of time after bud emergence. Although a key cellular function of these two formins in yeast is to promote actin cable formation (8, 18), the roles of the individual formins in different cellular process is unclear because deleting either individual formin gene has limited impact on cell growth and deleting both genes together is lethal (21).Although each of the two formins can nucleate actin filament formation in vitro, the manner in which they affect polymerization is distinctly isoform-specific. Most of this mechanistic work in vitro has used formin fragments containing the FH1 and FH2 domains. Bni1 alone processively caps the barbed end of actin filaments partially inhibiting polymerization at this end (14, 16, 18). The profilin-actin complex, recruited to the actin barbed end through its binding to Bni1 FH1 domain, possibly raises the local actin concentration and appears to allow this inhibition to be overcome, thereby, accelerating barbed end polymerization. It has also been shown that this complex modifies the kinetics of actin dynamics at the barbed end (9, 11, 18, 22). Moreover, Bni1 participation leads only to the formation of single filaments (8). In comparison, the Bnr1 FH1-FH2 domain facilitates actin polymerization much more efficiently than does Bni1. Moseley and Goode (8) showed Bnr1 accelerates actin polymerization up to 10 times better than does Bni and produces actin filament bundles when the Bnr1/actin molar ratio is above 1:2. Finally, the regulation of Bni1 and Bnr1 by formin binding is different. For example, Bud 6/Aip3, a yeast cell polarity factor, binds to Bni1, but not Bnr1, and also stimulates its activity in vitro.For their studies, Moseley and Goode (8) utilized mammalian skeletal muscle actin instead of the S. cerevisiae actin with which the yeast formins are designed to function. It is entirely possible that the differences observed with the two formins are influenced quantitatively or qualitatively by the nature of the actin used in the study. This possibility must be seriously considered because although yeast and muscle actins are 87% identical in sequence, they display marked differences in their polymerization behavior (23). Yeast actin nucleates filaments better than muscle actin (24, 25). It appears to form shorter and more flexible filaments than does muscle actin (26, 27). Finally, the disposition of the P_i released during the hydrolysis of ATP that occurs during polymerization is different. Yeast actin releases its P_i concomitant with hydrolysis of the bound ATP whereas muscle actin retains the P_i for a significant amount of time following nucleotide hydrolysis (28, 29). This difference is significant because ADP-P_i F-actin has been shown to be more stable than ADP F-actin (30). Another example of this isoform dependence is the interaction of yeast Arp2/3 with yeast versus muscle actins (31). Yeast Arp2/3 complex accelerates polymerization of muscle actin only in the presence of a nucleation protein factor such as WASP. However, with yeast actin, no such auxiliary protein is required. In light of these actin behavioral differences, to better understand the functional differences of these two formins in vivo, we have studied the behavior of Bni 1 and Bnr 1 with WT and mutant yeast actins, and we have also explored the molecular basis underlying the Bnr 1-induced formation of actin nuclei from G-actin.     

转录因子Oct-4调控下游靶基因及其与胚胎发育全能/多能性的关系

作为一个和胚胎发育全能／多能性相关的转录因子,Oct-4通过多种多样的调控机制激活或抑制不同靶基因的转录,从而在细胞的全能／多能性及未分化状态的调控维持中发挥重要的作用。已知受Oct-4调控的靶基因中,不仅有一些重要的转录因子如Rex-I,而且有一些参与重要细胞活动的基因如Fgf-4,因此对Oct-4调控下游靶基因的研究将有助于对其在分化发育中所起作用的进一步了解,同时对全能／多能性这一发育学基本问题及其调控网络有一个新的认识。     

Differences in the Regulation of K-Ras and H-Ras Isoforms by Monoubiquitination

Ras GTPases are signaling switches that control critical cellular processes including gene expression, differentiation, and apoptosis. The major Ras isoforms (K, H, and N) contain a conserved core GTPase domain, but have distinct biological functions. Among the three Ras isoforms there are clear differences in post-translational regulation, which contribute to differences in localization and signaling output. Modification by ubiquitination was recently reported to activate Ras signaling in cells, but the mechanisms of activation are not well understood. Here, we show that H-Ras is activated by monoubiquitination and that ubiquitination at Lys-117 accelerates intrinsic nucleotide exchange, thereby promoting GTP loading. This mechanism of Ras activation is distinct from K-Ras monoubiquitination at Lys-147, which leads to impaired regulator-mediated GTP hydrolysis. These findings reveal that different Ras isoforms are monoubiquitinated at distinct sites, with distinct mechanisms of action, but with a common ability to chronically activate the protein in the absence of a receptor signal or oncogenic mutation.     

植物类黄酮生物合成途径及重要基因的调控

类黄酮是一类广泛存在于植物中的多酚类次生代谢产物,具有抗氧化、抗炎、抗病毒、调节机体免疫力等多种功效,从而可以预防癌症、冠心病、中风等疾病,同时还具有抗衰老的作用.因此,类黄酮被人们称为"植物营养素".丰富的类黄酮种类和生物活性已使其成为世界保健品研究的热点和重点之一,本文比较详细地总结和阐述了类黄酮生物合成途径及重要基因的调控机制,使我们能够深入和全面地认识类黄酮,对从分子水平上研究和调节类黄酮的合成具有重要的意义.?＃