Novel transcriptome data analysis implicates circulating microRNAs in epigenetic inheritance in mammals

Experimental evidence supports a role of mobile small non-coding RNAs in mediating soma to germline hereditary information transfer in epigenetic inheritance in plants and worms. Similar evidence in mammals has not been reported so far. In this bioinformatic analysis, differentially expressed microRNAs (miRNAs) or mRNAs reported previously in genome level expression profiling studies related to or relevant in epigenetic inheritance in mammals were examined for circulating miRNA association. The reported sets of differentially expressed miRNAs or miRNAs that are known to target the reported sets of differentially expressed genes, in that order, showed enrichment of circulating miRNAs across environmental factors, tissues, life cycle stages, generations, genders and species. Circulating miRNAs commonly representing the expression profiles enriched various epigenetic processes. These results provide bioinformatic evidence for a role of circulating miRNAs in epigenetic inheritance in mammals.     

Expression of Alternative Ago2 Isoform Associated with Loss of microRNA-Driven Translational Repression in Mouse Oocytes

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Demonstrating polymorphic miRNA-mediated gene regulation in vivo: Application to the g+6223G→A mutation of Texel sheep

We herein describe the development of a biochemical method to evaluate the effect of single nucleotide polymorphisms (SNPs) in target genes on their regulation by microRNAs in vivo. The method is based on the detection of allelic imbalance in RNAs coimmunoprecipitated with AGO proteins from tissues of heterozygous individuals. We characterize the performances of our approach using a model system in a cell culture, and then apply it successfully to prove that the 3′UTR g+6223G→A mutation operates by promoting RISC-dependent down-regulation of myostatin (MSTN) in skeletal muscle of Texel sheep.     

FUS Regulates Activity of MicroRNA-Mediated Gene Silencing

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人类胚胎干细胞分化过程中DPPA2基因的表达情况分析(英文)

DPPA2(Developmental Pluripotency-Associated gene2)是近年来发现的在多能性细胞中特异表达的一个基因,它被认为参与维持干细胞的"干性".但目前为止,并没有关于该基因在人类胚胎干细胞(human embryonic stem cells,hESCs)分化过程中的表达情况的报道,其功能也尚不清楚.通过Real-time PCR对DPPA2基因在hESCs分化过程中的表达情况进行分析,此外还对其在异常核型hESCs,人类胚胎癌细胞(human embryonic carcinoma cells,hECCs)NTERA-2以及其它5种癌细胞中的表达情况进行检测.结果表明DPPA2基因在hESCs中特异表达,其表达水平随着hESCs的分化而显著下调.该基因在异常核型hESCs和NTERA-2细胞中也有表达,但在其它肿瘤细胞中未检测到该基因的表达.此外,以EGFP-N1系统为基础的亚细胞信号定位结果表明,DPPA2是一个核蛋白.这些结果提示,DPPA2基因可能在维持hESCs特性的过程中发挥着重要的作用.     

sTRAIL基因引起人恶性畸胎瘤细胞与人胚胎干细胞凋亡比较

目的:评估sTRAIL基因引起人胚胎干细胞[human embryonic stem(hES)cells]和人恶性畸胎瘤(human malignant teratoma)细胞NTERA-2凋亡的作用。方法:本室已构建的pAAV-PEG3-sTRAIL质粒,转染NTERA-2和hES细胞,用流式细胞仪和定量PCR检测NTERA-2和hES细胞的早期凋亡及sTRAIL基因的受体的表达。结果:NTERA-2细胞经瞬时转染pAAV-PEG3-sTRAIL质粒后,早期凋亡比例达24.4%±2.1%,sTRAIL的死亡受体DR4、和DR5的表达分别上升2和3倍,而hES细胞转染pAAV-PEG3-sTRAIL质粒后,早期凋亡比例仅2.3%±1.2%,受体DR4、DR5、DcR1和DcR2的表达未有明显变化。结论:pAAV-PEG3-sTRAIL质粒可有效地引起人恶性畸胎瘤细胞NTERA-2的凋亡,但是对人胚胎干细胞尚未发现可见的细胞凋亡现象。     

The novel MER transposon-derived miRNAs in human genome

MicroRNAs (miRNAs) are small RNA molecules (~ 20–30 nucleotides) that generally act in gene silencing and translational repression through the RNA interference pathway. They generally originate from intergenic genomic regions, but some are found in genomic regions that have been characterized such as introns, exons, and transposable elements (TE). To identify the miRNAs that are derived from palindromic MERs, we analyzed MER paralogs in human genome. The structures of the palindromic MERs were similar to the hairpin structure of miRNA in humans. Three miRNAs derived from MER96 located on chromosome 3, and MER91C paralogs located on chromosome 8 and chromosome 17 were identified in HeLa, HCT116, and HEK293 cell lines. The interactions between these MER-derived miRNAs and AGO1, AGO2, and AGO3 proteins were validated by immunoprecipitation assays. The data suggest that miRNAs derived from transposable elements could widely affect various target genes in the human genome.     

高效诱导人胚胎干细胞成为内皮祖细胞

血管的发生和发育不仅对胚胎形成中各器官的发育分化十分重要,并且对成体的创伤修复和生殖功能也具有重要意义．血管内皮细胞是形成心血管封闭管道系统的形态基础,体外多种细胞可经诱导分化产生出内皮祖/内皮细胞(endothelial progenitor/endothelial cells,EPCs/ECs),但是存在一些不足．鉴于人类胚胎干细胞(human embryonic stem cells,hESCs)诱导分化的全能性和长期增殖能力,为EPCs/ECs提供了新的来源．现有文献报道,hESCs诱导分化为EPCs/ECs的比例较低,为了提高该诱导分化效率,我们使用分阶段的二维诱导方法,首先将细胞接种在超纯层纤连蛋白(Matrigel)上,之后通过在不同阶段添加不同的因子,最终获得CD31⁺KDR⁺细胞的比例可以达到16%．进一步内皮诱导分化的结果显示,获得的EPCs/ECs的比例可以达到约32%,这些细胞具有在Matrigel上形成血管样结构的能力,可结合植物凝集素．实时定量PCR的结果显示,诱导分化所得的细胞表达众多内皮相关基因,并且免疫荧光的结果也表明部分细胞表达内皮细胞特异性表面标志CD31．     

Functional parameters of Dicer-independent microRNA biogenesis

Until recently, a Dicer-class RNase III enzyme was believed to be essential for microRNA (miRNA) biogenesis in all animals. The conserved vertebrate locus mir-451 defies this expectation and instead matures by direct cleavage of its pre-miRNA hairpin via the Slicer activity of Argonaute2 (Ago2). In this study, we used structure-function analysis to define the functional parameters of Ago2-mediated miRNA biogenesis. These include (1) the requirement for base-pairing at most, but not all, positions within the pre-mir-451 stem; (2) surprisingly little influence of the 5'-nucleotide on Ago sorting; (3) substantial influence of Ago protein stoichiometry on mir-451 maturation; (4) strong influence of G:C content in the distal stem on 3' resection of cleaved mir-451 substrates; and (5) the influence of hairpin length on substrate utilization by Ago2 and Dicer. Unexpectedly, we find that certain hairpin lengths confer competence to mature via both Dicer-mediated and Ago2-mediated pathways, and we show, in fact, that a conventional shRNA can traverse the Dicer-independent pathway. Altogether, these data inform the design of effective Dicer-independent substrates for gene silencing and reveal novel aspects of substrate handling by Ago proteins.     

PIWIL1在不同肿瘤细胞中的差异性表达

PIWI作为AGO蛋白家族的成员,在睾丸中特异表达,在精子形成过程中扮演着重要角色.已有文献报道,PIWIL2也广泛存在于多种肿瘤中,与肿瘤的发生相关.本文旨在确定PIWL1在不同肿瘤细胞中的表达差异性,进而初步探讨PIWIL1的表达差异与肿瘤发生发展的关系.半定量RT-PCR和Western 印迹检测多种肿瘤细胞中,PIWIL1在mRNA水平及蛋白水平的表达情况,进一步用免疫组化和免疫荧光检测PIWIL1在细胞中的表达及定位.PIWIL1在多种肿瘤细胞中的表达存在差异,其中一些肿瘤细胞中的表达水平较高,包括卵巢癌,前列腺癌,肝癌,胃癌等细胞.对于肿瘤细胞而言,PIWIL1定位于细胞浆中.因此,PIWIL1在多种肿瘤细胞中的表达差异性可能为肿瘤发生发展的研究提供新的线索.