鸡miR-148a的组织表达及其发育调控功能预测分析

为了解鸡miR-148a组织表达谱和潜在发育调控功能,采用茎-环定量RT-PCR检测了固始鸡5个发育阶段、15种组织中miR-148a的表达,利用Pictar和TargetScan算法预测了miR-148a的靶基因,并对预测靶基因分别进行了Gene Ontology分析和通路分析. 结果显示,miR-148a的表达具有明显的时序特征,胚胎期各组织中的表达水平明显低于出壳后;miR-148a在出壳后的大脑、小脑、延脑、腺胃、小肠、肝、胰和胸肌等器官组织中的表达水平随着发育进程被显著上调;miR-148a预测靶基因在胚胎器官形态发生、血细胞生成、消化道形态发生、心血管发育、感觉器官发育、肠发育、骨骼系统发育、后脑发育、呼吸系统发育、免疫系统发育、淋巴器官等发育过程显著富集. 总之,鸡miR-148a为广泛性表达miRNA,可能参与鸡诸多器官组织发育过程的调控.     

鸡miR-9不同组织表达差异及其功能预测分析

miRNA在动物生长发育过程中有重要作用. 本文采用定制茎环反转录引物,利用实时荧光定量PCR技术构建miR-9在鸡2个阶段11个组织中的表达谱,同时用TargetScan5.1与PicTar两种计算方法对其进行靶基因预测,交集的基因集合分别进行GO（gene ontology）富集分析和生物通路富集分析. 结果表明,采用实时定量PCR检测的miR-9在鸡下丘脑中的表达量和高通量测序结果一致;采用实时定量PCR在对不同组织定量结果表明,miR-9在0日龄鸡的肾脏、下丘脑、腿肌和大脑中高丰度表达,在成年鸡表达量较高为大脑、腿肌、心脏、小脑和下丘脑.在同一组织的0日龄和成年鸡中表达呈现时序性,除肝脏的表达量差异不显著,其他10个组织miR-9表达量差异显著（P<0.05）.预测到交集靶基因有160个.涉及到多个KEGG通路和GO富集中.GO分类结果显示,这些基因分布于63个群中,其中基因超过45个基因群集有26个群,与代谢有关的群有11个. 其它与发育、调控等过程有关. 在KEGG通路分析中,显著的通路有细胞骨架调控、细胞增殖和分化有关的通路（P<0.01）等5个通路. 表明miR 9基因的表达有组织和时序特异性,靶基因参与细胞代谢、生长发育和调控.这些结果为进一步验证miR 9基因在在脑中调控生长发育过程中的作用奠定了基础.     

牦牛miR-378前体克隆及组织表达分析

旨在克隆牦牛miR-378的前体序列,阐明其组织表达规律,结合bta-miR-378靶基因的生物信息学预测和分析,探讨miR-378在牦牛生长发育过程中的调控功能。采用PCR方法成功克隆类乌齐牦牛miR-378前体序列,实时荧光定量PCR(RT-qPCR)检测miR-378-3p在各组织中的表达模式,结合生物信息学软件TargetScan、DAVID以及数据库NCBI、miRbase等对miR-378进行保守性分析、靶基因预测及其生物学功能分析。结果表明,miR-378在各物种间高度保守,且miR-378-3p在各组织中广泛表达,其中在臀大肌中表达水平最高,显著高于其他组织(P<0.01),在臀脂中的表达高于卵巢、大脑、乳腺和肝脏。获得的272个靶基因主要参与细胞分化、细胞发育、大分子代谢等多个生物学过程,涉及孕酮介导的卵母细胞成熟、促性腺激素释放激素(GnRH)信号通路等,由此推测,miR-378可能在卵泡发育、卵母细胞成熟过程中起关键作用,进而影响母牦牛的繁殖性能。     

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。     

牦牛miR-383的靶基因预测及生物信息学分析

MicroRNAs (miRNAs)是一类非编码的小分子单链RNA,通过与靶基因的mRNA结合,抑制mRNA的翻译,参与多种生物学过程.本实验室前期通过高通量测序发现90日龄牦牛胚胎的背最长肌中miR-383的表达量显著高于成年牦牛.为探究miR-383在牦牛骨骼肌发育中的分子功能和机制,本研究对miR-383的靶基因进行预测,并进行生物信息学分析.通过TargetScan、miRDB和miRanda 3个软件预测了miR-383的靶基因,然后合并miRTarbase数据库中已被证实的靶基因作为基因集,分别用DAVID和KOBAS3.0在线软件对基因集进行功能注释(GO分析)和Pathway信号通路富集分析.结果 表明,牦牛miR-383序列在各物种间高度保守,靶基因功能富集于CD8阳性T细胞增殖的调控、核糖核蛋白复合物定位和负调控T细胞分化等生物学过程.信号通路分析发现靶基因的信号通路显著富集于PI3K-Ak、AMPK、FoxO和Focal adhesion等与肌肉发育相关的信号通路中.该研究结果将为miR-383功能及调控机制的深入研究提供参考依据,也为解析牦牛肌肉发育的分子机制提供新的研究方向.     

固始鸡1日龄和36周龄下丘脑高丰度差异性MiRNA的鉴定及功能预测分析

为鉴定鸡下丘脑发育相关特异性表达miRNA,基于固始鸡1日龄和36周龄下丘脑小RNA的Solexa测序数据,共鉴定到266种2个发育阶段共表达的miRNA,其中157种miRNA的表达水平被显著下调,22种被显著上调.聚类分析显示,鸡下丘脑高丰度差异性miRNA主要集中于let-7、mir-181、mir-30、mir-99、mir-1和mir-17等基因家族.另外,预测了10种高丰度差异性miRNA的靶基因,并进行了相应的GO分析和KEGG通路分析.结果显示,预测靶基因在发育过程、代谢过程、细胞过程和生物学过程调节等4个生物学过程以及细胞周期、粘着斑、TGF-beta信号通路和MAPK信号通路等通路中显著富集.研究结果为进一步揭示miRNA调控鸡下丘脑发育的分子机制提供了有益线索.     

Hsa-miR-10a-5p靶基因预测及生物信息学分析

利用生物信息学对miR-10a-5p的靶基因进行预测及相关分析,为miR-10a-5p靶基因的实验验证及其调控机制提供理论基础。通过miRBase获取并分析人、大鼠,小鼠等物种的miR-10a-5p的碱基序列特征;使用在线数据库Targetscan7.1,miRDB,mirDIP和DIANA TOOLS等预测miR-10a-5p的靶基因,并用Venny 2.1绘制韦恩图取交集。用在线工具DAVID对交集靶基因进行GO功能注释和KEGG pathway分析。结果表明miR-10a-5p成熟序列在各物种间高度保守。获得的79个靶基因在分子功能上主要涉及染色质结合,转录活性激活等,并显著富集于肝脏发育,脂肪组织发育等生物学过程,涉及cAMP信号通路,TNF信号通路及AMPK信号通路。miR-10a-5p通过调控靶基因参与了生命活动和疾病过程中多个方面,尤其生长发育和癌症过程,为进一步研究提供了生物学基础。     

miR-143在脂肪细胞分化和脂类代谢中的作用

miR-143是内源性非编码的RNA,在细胞的分化、发育、增殖、脂肪代谢及疾病产生等许多生物学过程中具有重要作用。miR-143与脂类代谢密切相关,主要通过与靶基因的相互作用,影响脂肪的分化,甘油三酯的合成等。针对miR-143合成、预测的靶基因以及靶基因的调控机制方面进行综述,阐述miR-143在脂肪代谢中的作用,以便为相关疾病的诊断、治疗方面提供新思路。     

miR-133b可能通过调控foxO1的表达影响小鼠B细胞发育

microRNAs(miRNAs)是一类具有转录后调控作用的非编码RNA,在发育、细胞增殖、凋亡及肿瘤发生等多种生理和病理过程中发挥重要作用.为全面了解小鼠B细胞中miRNAs的表达模式,利用流式细胞仪(FACS)分选处于不同发育时期的B细胞,采用TaqMan誖低密度芯片对其进行检测,筛选到pre-B阶段9个miRNAs表达量显著上调.将筛选出的miRNAs进行靶基因预测,并对预测靶基因进行功能聚类和通路分析,发现约4%的基因参与免疫系统过程,包括Bcl2、Kit等.选取foxO1与miR-19b、miR-142-3p、miR-106b、miR-182及miR-133b进行初步功能验证,双荧光素酶报告系统及Westernblot检测结果均显示,miR-133b可直接作用于foxO1 3′UTR从而降低foxO1的表达.结合人类和小鼠B细胞中foxO1的表达情况分析,其表达模式同miR-133b表达模式呈负相关,说明miR-133b可能参与了B细胞发育过程中foxO1的表达调控过程.     

miR-24-3p对宫颈癌细胞增殖与迁移的作用及机制研究

该文探讨了miR-24-3p对宫颈癌细胞增殖和迁移的促进作用及其机制。采用miR-24-3p抑制剂下调宫颈癌细胞中miR-24-3p的表达后,通过MTT、Transwell实验和Western blot检测细胞增殖、迁移和PCNA蛋白水平;采用生物信息学方法预测miR-24-3p的靶基因并进行功能注释和筛选;双荧光素酶报告实验和Western blot验证靶基因类血管动蛋白-2(angiomotin-like 2,AMOTL2),并通过siRNA抑制AMOTL2检测其对宫颈癌细胞迁移的影响。结果显示,下调miR-24-3p能抑制宫颈癌细胞的增殖和迁移能力并减少PCNA蛋白表达;其靶基因主要存在细胞与细胞连接组分中,显著富集于蛋白激酶活性分子功能、蛋白质自身磷酸化生物学过程和癌症中microRNA信号通路;miR-24-3p能靶向负调控最佳靶基因AMOTL2,下调AMOTL2可促进宫颈癌细胞CaSki的迁移。总之,miR-24-3p可调控多靶基因参与多个生物学过程和多条信号通路,在宫颈癌中可促进细胞增殖且通过靶向AMOTL2促进迁移。     

Differential Toll-Like Receptor (TLR) mRNA Expression Patterns during Chicken Embryological Development

Toll-like receptors (TLRs), important components of innate immune response, play a pivotal role in early recognition of pathogen as well as in the initiation of robust and specific adaptive immune response. In the present study, the expression profile of chicken TLRs (TLR2A, TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21) in various chicken embryonic tissues during embryo development was examined by real-time PCR assay. All the TLR mRNAs were expressed in whole embryonic tissue as early as 3rd embryonic day (ED). Four of the seven TLRs (TLR2, TLR3, TLR4, and TLR7) mRNA expressions were significantly (P < 0.01) higher at 12ED relative to expression at 3 ED, whereas TLR15 mRNA expression was significantly (P < 0.01) higher on 7ED and TLR5 and 21 were highly expressed on 18 ED. Among all the TLRs investigated TLR4 mRNA was the highest expressed and TLR15 mRNA expression was the lowest in all tissues during chicken embryo development. Tissue wise analysis of mRNA expression of TLRs showed that liver expressed significantly (P < 0.01) higher levels of most of the genes (TLR2, TLR4, and TLR21). However no significant difference was found in TLR15 mRNA expression among the tissues during development. Our results suggest the innate preparedness of chicken embryos and also a possible role for TLRs in the regulation of chicken embryo development that needs to be further evaluated.     

Cloning and expression analysis of the chicken DEAD box gene DDX1

DEAD box proteins are putative RNA unwinding proteins found in organisms ranging from mammals to bacteria. While some DEAD box genes expressed in higher eukaryotes are ubiquitous, others have distribution profiles that suggest a cell-, tissue-, or developmental-specific role. The DEAD box gene, DDX1, was identified by differential screening of a subtracted retinoblastoma cDNA library. A limited survey of human fetal tissues indicated that DDX1 mRNA has a widespread distribution but is not uniformly expressed in all tissues. To further document the spatial and temporal distribution of DDX1 during embryonic development, we cloned the chicken DDX1 cDNA. The predicted amino acid sequence of chicken DDX1 was 93% identical to that of human DDX1. All DEAD box motifs, as well as a SPRY domain, were present in chicken DDX1. Northern and Western blot analyses showed highest levels of DDX1 at early stages of development. Tissue maturation was generally accompanied by a decrease in expression, although DDX1 levels remained elevated in late embryonic retina and brain. In situ hybridization of retinal tissue sections revealed widespread distribution of DDX1 mRNA at early developmental stages with preferential expression in amacrine and ganglion cells of the differentiated tissue. Preferential expression of DDX1 was also observed in specific areas of the brain in older embryos, such as the external granule layer of the cerebellum. These results suggest a specific role for DDX1 in subsets of differentiated cells as well as a more general role in undifferentiated cells.     

Fatty acid synthesis in chick embryonic heart and liver during development.

Fatty acid synthesis by subcellular fractions of heart and liver of chick embryos at varying stages of development has been studied. Fatty acid synthetase activity is associated with the embryonic heart at early stages of development, as suggested by substrate requirement, Schmidt decarboxylation of synthesized fatty acids and gas liquid chromatographic identification of the products as palmitic and stearic acids. The fatty acid synthetase activity decreases in heart cytosol with age of the embryo and is absent in the newly hatched chick and in older chicken. The acetyl CoA carboxylase activity is negligible in embryonic and adult chicken heart. The fatty acid synthetase activity in liver is low, but measurable during the entire embryonic development. The activity increases by about three-fold on hatching and thereafter in fed, newly hatched chicks by about 35-fold, over the basal embryonic activity. The acetyl and malonyl transacylase activities in the heart and liver cytosols during development followed closely the fatty acid synthetase activities in heart and liver, respectively. A non-coordinate induction of fatty acid synthetase and acetyl CoA carboxylase activities in liver was observed during development. The microsomal chain elongation in liver and heart followed the pattern of fatty acid synthetase activity in liver and heart, respectively. The mitochondrial chain elongation in embryonic heart is initially low and increases with age; while this activity in liver is higher in early stages of embryonic development than in the older embryos and the chicks. Measurement of lipogenesis from acetate-1-¹⁴C by liver and heart slices from chick embryos and newly hatched chicks support the conclusions reached in the studies with the subcellular fractions. The results obtained indicate that the major system of fatty acid synthesis in embryonic and adult heart is the mitochondrial chain elongation. In embryonic liver, fatty acid synthesis proceeds by chain elongation, while the de novo system is the major contributor to the lipogenic capacity of the liver after hatching.     

Developmental increase in the inotropic and cyclic AMP response to isoproterenol in embryonic and newly hatched chicks

The cyclic adenosine 3',5'-monophosphate (cyclic AMP) levels of ventricles isolated from 15- to 20-day-old chick embryos and 0- to 3-day-old hatched chicks were compared to clarify the mechanism underlying the change in sensitivity to isoproterenol during perinatal developmental stages when the functional sympathetic innervation has been completely achieved. Isoproterenol produced a positive inotropic effect on ventricles isolated from both embryonic and hatched chicks, but the ventricles from the hatched chicks were more sensitive. At both developmental stages sotalol was an equipotent antagonist of isoproterenol. 3-Isobutyl-1-methylxanthine (IBMX) produced an increment in the contractile force of the ventricles at both stages, but the ventricles from the hatched chicks responded to lower doses of IBMX. The reactivity to isoproterenol in increasing cyclic AMP level was significantly higher in the hatched ventricles than in the embryonic ventricles. The results suggest that the different sensitivities to isoproterenol between embryonic and newly hatched chick ventricles may be due to some changes in the process for cyclic AMP production.