星形细胞瘤生存预后相关miRNA-mRNA调控关系对筛选

利用GEO数据库中的芯片数据,筛选与星形细胞瘤生存预后相关的miRNA-mRNA调控关系对,为后续研究提供理论支持.下载芯片数据利用R语言进行差异表达分析,得到星形细胞瘤较正常组织表达显著改变的miRNA与mRNA;通过miRNA靶基因预测,将靶基因与差异表达mRNA取交集,明确mRNA与miRNA之间的关系;利用GE...     

Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells

Kaposi’s sarcoma associated herpesvirus (KSHV) causes several tumors, including primary effusion lymphoma (PEL) and Kaposi’s sarcoma (KS). Cellular and viral microRNAs (miRNAs) have been shown to play important roles in regulating gene expression. A better knowledge of the miRNA-mediated pathways affected by KSHV infection is therefore important for understanding viral infection and tumor pathogenesis. In this study, we used deep sequencing to analyze miRNA and cellular mRNA expression in a cell line with latent KSHV infection (SLKK) as compared to the uninfected SLK line. This approach revealed 153 differentially expressed human miRNAs, eight of which were independently confirmed by qRT-PCR. KSHV infection led to the dysregulation of ~15% of the human miRNA pool and most of these cellular miRNAs were down-regulated, including nearly all members of the 14q32 miRNA cluster, a genomic locus linked to cancer and that is deleted in a number of PEL cell lines. Furthermore, we identified 48 miRNAs that were associated with a total of 1,117 predicted or experimentally validated target mRNAs; of these mRNAs, a majority (73%) were inversely correlated to expression changes of their respective miRNAs, suggesting miRNA-mediated silencing mechanisms were involved in a number of these alterations. Several dysregulated miRNA-mRNA pairs may facilitate KSHV infection or tumor formation, such as up-regulated miR-708-5p, associated with a decrease in pro-apoptotic caspase-2 and leukemia inhibitory factor LIF, or down-regulated miR-409-5p, associated with an increase in the p53-inhibitor MDM2. Transfection of miRNA mimics provided further evidence that changes in miRNAs are driving some observed mRNA changes. Using filtered datasets, we also identified several canonical pathways that were significantly enriched in differentially expressed miRNA-mRNA pairs, such as the epithelial-to-mesenchymal transition and the interleukin-8 signaling pathways. Overall, our data provide a more detailed understanding of KSHV latency and guide further studies of the biological significance of these changes.     

Discovery of microRNA-mRNA modules via population-based probabilistic learning

MOTIVATION: MicroRNAs (miRNAs) and mRNAs constitute an important part of gene regulatory networks, influencing diverse biological phenomena. Elucidating closely related miRNAs and mRNAs can be an essential first step towards the discovery of their combinatorial effects on different cellular states. Here, we propose a probabilistic learning method to identify synergistic miRNAs involving regulation of their condition-specific target genes (mRNAs) from multiple information sources, i.e. computationally predicted target genes of miRNAs and their respective expression profiles. RESULTS: We used data sets consisting of miRNA-target gene binding information and expression profiles of miRNAs and mRNAs on human cancer samples. Our method allowed us to detect functionally correlated miRNA-mRNA modules involved in specific biological processes from multiple data sources by using a balanced fitness function and efficient searching over multiple populations. The proposed algorithm found two miRNA-mRNA modules, highly correlated with respect to their expression and biological function. Moreover, the mRNAs included in the same module showed much higher correlations when the related miRNAs were highly expressed, demonstrating our method's ability for finding coherent miRNA-mRNA modules. Most members of these modules have been reported to be closely related with cancer. Consequently, our method can provide a primary source of miRNA and target sets presumed to constitute closely related parts of gene regulatory pathways.     

Large-Scale Screens of miRNA-mRNA Interactions Unveiled That the 3′UTR of a Gene Is Targeted by Multiple miRNAs

Animal microRNA (miRNA) target prediction is still a challenge, although many prediction programs have been exploited. MiRNAs exert their function through partially binding the messenger RNAs (mRNAs; likely at 3′ untranslated regions [3′UTRs]), which makes it possible to detect the miRNA-mRNA interactions in vitro by co-transfection of miRNA and a luciferase reporter gene containing the target mRNA fragment into mammalian cells under a dual-luciferase assay system. Here, we constructed a human miRNA expression library and used a dual-luciferase assay system to perform large-scale screens of interactions between miRNAs and the 3′UTRs of seven genes, which included more than 3,000 interactions with triplicate experiments for each interaction. The screening results showed that the 3′UTR of one gene can be targeted by multiple miRNAs. Among the prediction algorithms, a Bayesian phylogenetic miRNA target identification algorithm and a support vector machine (SVM) presented a relatively better performance (27% for EIMMo and 24.7% for miRDB) against the average precision (17.3%) of the nine prediction programs used here. Additionally, we noticed that a relatively high conservation level was shown at the miRNA 3′ end targeted regions, as well as the 5′ end (seed region) binding sites.     

MicroRNA-21 Regulates PI3K/Akt/mTOR Signaling by Targeting TGFβI during Skeletal Muscle Development in Pigs

MicroRNAs (miRNAs), which are short (22–24 base pairs), non-coding RNAs, play critical roles in myogenesis. Using Solexa deep sequencing, we detected the expression levels of 229 and 209 miRNAs in swine skeletal muscle at 90 days post-coitus (E90) and 100 days postnatal (D100), respectively. A total of 138 miRNAs were up-regulated on E90, and 31 were up-regulated on D100. Of these, 9 miRNAs were selected for the validation of the small RNA libraries by quantitative RT-PCR (RT-qPCR). We found that miRNA-21 was down-regulated by 17-fold on D100 (P<0.001). Bioinformatics analysis suggested that the transforming growth factor beta-induced (TGFβI) gene was a potential target of miRNA-21. Both dual luciferase reporter assays and western blotting demonstrated that the TGFβI gene was regulated by miRNA-21. Co-expression analysis revealed that the mRNA expression levels of miRNA-21 and TGFβI were negatively correlated (r = -0.421, P = 0.026) in skeletal muscle during the 28 developmental stages. Our results revealed that more miRNAs are expressed in prenatal than in postnatal skeletal muscle. The miRNA-21 is a novel myogenic miRNA that is involved in skeletal muscle development and regulates PI3K/Akt/mTOR signaling by targeting the TGFβI gene.     

H-Ferritin-Regulated MicroRNAs Modulate Gene Expression in K562 Cells

In a previous study, we showed that the silencing of the heavy subunit (FHC) offerritin, the central iron storage molecule in the cell, is accompanied by a modification in global gene expression. In this work, we explored whether different FHC amounts might modulate miRNA expression levels in K562 cells and studied the impact of miRNAs in gene expression profile modifications. To this aim, we performed a miRNA-mRNA integrative analysis in K562 silenced for FHC (K562^shFHC) comparing it with K562 transduced with scrambled RNA (K562^shRNA). Four miRNAs, namely hsa-let-7g, hsa-let-7f, hsa-let-7i and hsa-miR-125b, were significantly up-regulated in silenced cells. The remarkable down-regulation of these miRNAs, following FHC expression rescue, supports a specific relation between FHC silencing and miRNA-modulation. The integration of target predictions with miRNA and gene expression profiles led to the identification of a regulatory network which includes the miRNAs up-regulated by FHC silencing, as well as91 down-regulated putative target genes. These genes were further classified in 9 networks; the highest scoring network, “Cell Death and Survival, Hematological System Development and Function, Hematopoiesis”, is composed by 18 focus molecules including RAF1 and ERK1/2. We confirmed that, following FHC silencing, ERK1/2 phosphorylation is severely impaired and that RAF1 mRNA is significantly down-regulated. Taken all together, our data indicate that, in our experimental model, FHC silencing may affect RAF1/pERK1/2 levels through the modulation of a specific set of miRNAs and add new insights in to the relationship among iron homeostasis and miRNAs.     

Joint Genome-Wide Profiling of miRNA and mRNA Expression in Alzheimer's Disease Cortex Reveals Altered miRNA Regulation

Although microRNAs are being extensively studied for their involvement in cancer and development, little is known about their roles in Alzheimer''s disease (AD). In this study, we used microarrays for the first joint profiling and analysis of miRNAs and mRNAs expression in brain cortex from AD and age-matched control subjects. These data provided the unique opportunity to study the relationship between miRNA and mRNA expression in normal and AD brains. Using a non-parametric analysis, we showed that the levels of many miRNAs can be either positively or negatively correlated with those of their target mRNAs. Comparative analysis with independent cancer datasets showed that such miRNA-mRNA expression correlations are not static, but rather context-dependent. Subsequently, we identified a large set of miRNA-mRNA associations that are changed in AD versus control, highlighting AD-specific changes in the miRNA regulatory system. Our results demonstrate a robust relationship between the levels of miRNAs and those of their targets in the brain. This has implications in the study of the molecular pathology of AD, as well as miRNA biology in general.     

n-3多不饱和脂肪酸调节饮食诱导肥胖大鼠miRNA表达变化(英文)

目的:研究n-3多不饱和脂肪酸(polyunsaturated fatty acids,PUFA)饮食对饮食诱导肥胖大鼠的miR NA表达影响。方法:将10只饮食诱导肥胖(diet induced obese,DIO)大鼠随机分成两组:n-3PUFA添加组和安慰剂添加组(对照组);每周记录两组老鼠的体重、体长和进食量。对外周血miR NA的表达并进行分析和预测。结果:两组老鼠Lee指数有统计学差异(P0.05);与对照组相比,在n-3组的外周血单核细胞中,29个miR NA上调,31个下调;其中rno-miR-200和rno-miR-211的表达量上调,rno-miR-29b和rno-miR-92b的表达量下调,其靶基因预测结果与神经营养因子,脂肪细胞因子,趋化因子和胰岛素信号通路有关。结论:n-3PUFA能够调节DIO大鼠的miR NA水平,其中有些与脂肪代谢相关。     

Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic, Androgenetic and Fertilized Blastocysts

MicroRNAs (miRNAs) are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions. In this study, miRNA and messenger RNA (mRNA) profiles were examined by Illumina microarray in mouse embryonic stem cells (ESCs) derived from parthenogenetic, androgenetic, and fertilized blastocysts. The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression. Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs (aESCs) and fertilized ESCs (fESCs), a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs (pESCs) and fESCs, and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs. In addition, a total of 575, 5 and 376 miRNA-mRNA target pairs were observed in aESCs vs. fESCs, pESCs vs. fESCs, and aESCs vs. pESCs, respectively. Furthermore, 15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR. Finally, transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3, Dyrk1b, and Prrg2 mRNA, inhibition of miR-363 increased the expression of Nfat5 and Soat1 mRNA, and inhibition of miR-883b-5p increased Nfat5, Tacstd2, and Ppapdc1 mRNA. These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.