Silencing of microRNAs in vivo with 'antagomirs'

MicroRNAs (miRNAs) are an abundant class of non-coding RNAs that are believed to be important in many biological processes through regulation of gene expression. The precise molecular function of miRNAs in mammals is largely unknown and a better understanding will require loss-of-function studies in vivo. Here we show that a novel class of chemically engineered oligonucleotides, termed 'antagomirs', are efficient and specific silencers of endogenous miRNAs in mice. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 resulted in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals. The silencing of endogenous miRNAs by this novel method is specific, efficient and long-lasting. The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the 3' untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. Our findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in disease.     

美洲鲎miRNA的生物信息学挖掘与分析

利用miRBase数据库中已有动物的小RNA（miRNA）,通过生物信息学方法对美洲鲎(Limulus polyphemus)表达序列标签（Expressed Sequences Tag,EST）和cDNA进行序列比对,挖掘美洲鲎miRNA,得到了 72个miRNA前体(pre-miRNA)、49个可编码成熟miRNA,隶属于40个miRNA家族。miRNA家族归类结果表明,无论在低等脊椎动物还是高等动物中,miRNA家族的存在都是相当保守的。以miR-10基因前体为例,分析其序列与其他物种同源序列的差异,显示miRNA序列的高度保守性。miR-10前体序列分析系统进化发现,物种聚类与传统分类亲缘关系一致。     

A pancreatic islet-specific microRNA regulates insulin secretion

MicroRNAs (miRNAs) constitute a growing class of non-coding RNAs that are thought to regulate gene expression by translational repression. Several miRNAs in animals exhibit tissue-specific or developmental-stage-specific expression, indicating that they could play important roles in many biological processes. To study the role of miRNAs in pancreatic endocrine cells we cloned and identified a novel, evolutionarily conserved and islet-specific miRNA (miR-375). Here we show that overexpression of miR-375 suppressed glucose-induced insulin secretion, and conversely, inhibition of endogenous miR-375 function enhanced insulin secretion. The mechanism by which secretion is modified by miR-375 is independent of changes in glucose metabolism or intracellular Ca2+-signalling but correlated with a direct effect on insulin exocytosis. Myotrophin (Mtpn) was predicted to be and validated as a target of miR-375. Inhibition of Mtpn by small interfering (si)RNA mimicked the effects of miR-375 on glucose-stimulated insulin secretion and exocytosis. Thus, miR-375 is a regulator of insulin secretion and may thereby constitute a novel pharmacological target for the treatment of diabetes.     

Regulation of microRNA on plant development and viral infection

THE FIRST MIRNA WAS IDENTIFIED IN C. ELEGANS AS EARLY AS IN 1993; THE IMPORTANCE OF MIRNAS, HOWEVER, IS RECOGNIZED ONLY RECENTLY AFTER THE DISCOVERY OF MIRNAS EXISTING UNIVERSALLY IN EUKARYOTIC ORGANISMS. THE SECOND MIRNA WAS IDENTIFIED IN 2000[1]. SINCE …     

Interferon modulation of cellular microRNAs as an antiviral mechanism

RNA interference through non-coding microRNAs (miRNAs) represents a vital component of the innate antiviral immune response in plants and invertebrate animals; however, a role for cellular miRNAs in the defence against viral infection in mammalian organisms has thus far remained elusive. Here we show that interferon beta (IFNbeta) rapidly modulates the expression of numerous cellular miRNAs, and that eight of these IFNbeta-induced miRNAs have sequence-predicted targets within the hepatitis C virus (HCV) genomic RNA. The introduction of synthetic miRNA-mimics corresponding to these IFNbeta-induced miRNAs reproduces the antiviral effects of IFNbeta on HCV replication and infection, whereas neutralization of these antiviral miRNAs with anti-miRNAs reduces the antiviral effects of IFNbeta against HCV. In addition, we demonstrate that IFNbeta treatment leads to a significant reduction in the expression of the liver-specific miR-122, an miRNA that has been previously shown to be essential for HCV replication. Therefore, our findings strongly support the notion that mammalian organisms too, through the interferon system, use cellular miRNAs to combat viral infections.     

RNAi-mediated gene silencing in non-human primates

The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (ApoB) in non-human primates. APOB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg(-1). A single siRNA injection resulted in dose-dependent silencing of APOB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of APOB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.     

Correlation of microRNAs responding to high dose γ-irradiation with predicted target mRNAs in HeLa cells using microarray analyses

An increasing data indicates that altered microRNAs （miRNAs） participate in the radiation-induced DNA damage response. However, a correlation of mRNA and miRNA profiles across the entire genome and in response to irradiation has not been thor- oughly assessed. We analyzed miRNA microarray data collected from HeLa cells after ionizing radiation （IR）, quantified the ex- pression profiles of mRNAs and performed comparative analysis of the data sets using target prediction algorithms, Gene Ontol- ogy （GO） analysis, pathway analysis, and gene network construction. The results showed that the altered miRNAs were involved in regulation of various cellular functions, miRNA-gene network analyses revealed that miR- 186, miR- 106b, miR- 15 a/b, CCND 1 and CDK6 played vital role in the cellular radiation response. Using qRT-PCR, we confirmed that twenty-two miRNAs showed differential expression in HeLa cells treated with IR and some of these miRNAs affected cell cycle progression. This study demonstrated that miRNAs influence gene expression in the entire genome during the cellular radiation response and suggested vital pathways for further research.     

血清miR-25、miR-223和miR-373作为食管鳞癌生物标志物的评价

为了探讨血清miRNA作为诊断标志物的可行性,利用茎环引物进行qRT-PCR,检测了miR-25、miR-223和miR-373在正常人血清、食管鳞癌患者术前和术后第7 d血清、癌组织和癌旁组织的相对表达量.实验结果表明:术前食管鳞癌病人的3种血清miRNA相对表达量高于正常人和手术后第7 d病人,AUC分别为0.794、0.839和0.873,癌组织的这3种miRNA相对表达量高于癌旁组织.这3种miRNA在食管癌患者癌组织的高表达导致血清的这3种miRNA含量增高,因此这3种血清miRNA可以作为候选诊断标志物.     

A viral microRNA functions as an orthologue of cellular miR-155

All metazoan eukaryotes express microRNAs (miRNAs), roughly 22-nucleotide regulatory RNAs that can repress the expression of messenger RNAs bearing complementary sequences. Several DNA viruses also express miRNAs in infected cells, suggesting a role in viral replication and pathogenesis. Although specific viral miRNAs have been shown to autoregulate viral mRNAs or downregulate cellular mRNAs, the function of most viral miRNAs remains unknown. Here we report that the miR-K12-11 miRNA encoded by Kaposi's-sarcoma-associated herpes virus (KSHV) shows significant homology to cellular miR-155, including the entire miRNA 'seed' region. Using a range of assays, we show that expression of physiological levels of miR-K12-11 or miR-155 results in the downregulation of an extensive set of common mRNA targets, including genes with known roles in cell growth regulation. Our findings indicate that viral miR-K12-11 functions as an orthologue of cellular miR-155 and probably evolved to exploit a pre-existing gene regulatory pathway in B cells. Moreover, the known aetiological role of miR-155 in B-cell transformation suggests that miR-K12-11 may contribute to the induction of KSHV-positive B-cell tumours in infected patients.     

酶标板上基于竞争杂交反应的几种miRNA序列的比色检测

多种miRNA序列的同时检测对癌症的预警与早期诊断具有重要的意义。本文基于生物素标记的miRNA和靶点miRNA与酶标板上固定的寡核苷酸探针之间的竞争反应,通过比色法来定量检测三种与胶质瘤相关的miRNA序列,即miR-182、miR-185和miR-381。通过亲和素与生物素之间的特异性相互作用,将亲和素标记的多聚辣根过氧化物酶(SA-polyHRP)衍生到微孔表面。通过检测HRP催化3,3′,5,5′-四甲基联苯胺和H2O2的显色反应来对三种靶点miRNA序列进行定量。方法最低检测浓度为10fmol/L,有望用于临床上miRNA的定量分析。碱基错配的结果表明该方法具有较好的选择性。     

循环血miR-21-3p作为脓毒症心肌病的标记物

已有研究发现微小RNA(microRNAs, miRNAs) 可由细胞分泌并释放进入循环系统, 在血清、血浆、尿液和其他体液中稳定存在, 并可作为非损伤性生物标记物对多种疾病进行早期诊断、鉴别分期、预后判断和疗效监测等. 与健康对照者相比, miR-21-3p 在22 例脓毒症心衰患者血浆中显著升高. 通过绘制受试者工作特征(receiver operating characteristic, ROC)曲线, 并分析其与临床参数的相关性发现, miR-21-3p 在鉴别脓毒症心衰和健康人群时, 曲线下面积(area under the curve, AUC)为0.949. 相关性分析结果显示, 血浆miR-21-3p水平与B 型脑钠肽(brain natriuretic peptide, BNP)(r = 0.968, P < 0.001)、心肌肌钙蛋白T (cardiac troponin T, cTNT)(r = 0.257, P = 0.002)水平呈正相关. 因此, 外周血中的miR-21-3p有作为脓毒症心肌病的生物标记物的潜力.     

藻蓝色素蛋白通过调控miR-642a-5p抑制LTEP-a2细胞的增殖和迁移

研究藻蓝色素蛋白抑制非小细胞肺癌LTEP-a2体外增殖迁移的机制.以LTEP-a2细胞为模型,采用高通量miRNA转录组学对藻蓝蛋白处理后细胞中差异表达的miRNA进行了筛选;对筛选出的差异miRNA,利用体外转染mimics的方法对细胞的增殖、迁移能力进行验证.研究结果显示,藻蓝蛋白处理LTEP-a2细胞后能够显著增加细胞内miR-642a-5p的表达水平;过表达miR-642a-5p能够显著抑制细胞的体外增殖、迁移能力;此外,藻蓝蛋白可以通过上调miR-642a-5p表达抑制核受体NF-κB信号通路,降低蛋白磷酸化水平,进而抑制LTEP-a2细胞的增殖迁移能力.研究能够为藻蓝色素蛋白的应用以及非小细胞肺癌的治疗提供一定的理论基础.