ABSTRACT: Kidney miRNA expression was examined in F344 rats at 2, 5, 6, 8, 15, 21, 78, and 104 weeks of age in both sexes using Agilent miRNA microarrays. 311 miRNAs were found to be expressed in at least one age and sex. Filtering criteria of ≥1.5 fold change and ANOVA (FDR <5%) revealed 174 differentially expressed miRNAs in the kidney; 173 and 34 miRNAs exhibiting age and sex effects, respectively. Principal component analysis revealed age effects predominated over sex effects, with 2 week miRNA expression being much different from other ages. No significant sexually dimorphic miRNA expression was observed from 5 to 8 weeks, while the most differential expression (13 miRNAs) was observed at 21 weeks. Potential target genes of these differentially expressed miRNAs were identified. Pathway analysis was used to investigate the possible roles of these target genes in age- and sex-specific differences. Untreated male and female F344 rats from 2, 5, 6, 8, 15, 21, 78, and 104 weeks of age (n=5) were sacrificed by CO2 asphyxiation, whole kidneys collected and homogenized, total RNA, including small RNA fraction, was used for miRNA expression arrays (Agilent).
Project description:Liver tissues from male and female F344 rats were examined over the life span for the purpose of evaluating global miRNAs with putative roles in drug metabolism and liver disease. Hepatic miRNA expression was characterized at 2, 5, 6, 8, 15, 21, 52, 78, and 104 weeks of age in both sexes using Agilent 8x15k rat miRNA microarrays containing multiple probes for 677 unique miRNAs. Data processing, 75% normalization, and statistical analysis of microarray data were performed in SAS. Functional analysis (Ingenuity) was performed on select miRNAs exhibiting prominent age and sex expression differences. + Untreated male and female F344 rats from 2, 5, 6, 8, 15, 21, 52, 78, and 104 weeks of age (n=5) were sacrificed by CO2 asphyxiation, whole livers collected and homogenized, total RNA, including small RNA fraction, was used for miRNA expression arrays (Agilent).
Project description:Global miRNA expression was examined in liver tissues from male and female F344 rats over the life span for the purpose of evaluating their possible roles in susceptibility to liver toxicity and disease. Hepatic miRNA expression was characterized at 2, 5, 6, 8, 15, 21, 52, 78, and 104 weeks of age in both sexes using Agilent 8x15k rat miRNA microarrays containing multiple probes for 677 unique miRNAs. Data processing, 75% normalization, and statistical analysis of microarray data were performed in SAS. Functional analysis (Ingenuity) was performed on select miRNAs exhibiting prominent age and sex expression differences. + Overall design: Untreated male and female F344 rats from 2, 5, 6, 8, 15, 21, 52, 78, and 104 weeks of age (n=5) were sacrificed by CO2 asphyxiation, whole livers collected and homogenized, total RNA, including small RNA fraction, was used for miRNA expression arrays (Agilent).
Project description:Drosophila miRNAs show distinct change in isoform distribution pattern with age. Some miRNAs show accumulation of the short isoforms, while other miRNAs show the accumulation of the long isoforms with age. The increase of the long isoforms of some miRNAs reflects increased 2'-O-methylated miRNA isoforms with age. This raised a question on whether additional miRNAs show increased 2'-O-methylated miRNA isoforms with age. To investigate change in 2'-O-methylated small RNA population with age, we performed small RNA deep-sequencing of oxidized or non-oxidized small RNAs at 3d and 30d in Drosophila. This analysis revealed change in global pattern of 2'-O-methylated miRNA isoforms with age. 3d and 30d wild-type male flies were collected. Total RNA was prepared with Trizol reagent, followed by oxidation/β-elimination assay or the control treatment (no-oxidation). Small RNA libraries were prepared using Illumina's TruSeq small RNA sample preparation kit (#RS-200-0012, Illumina, Inc. San Diego, CA), following the manufacturer's protocol. The libraries were multiplexed and sequenced on HiSeq2000 platform (Illumina).
Project description:Drosophila miRNAs show distinct change in isoform distribution pattern with age. Some miRNAs show accumulation of the short isoforms, while other miRNAs show the accumulation of the long isoforms with age. The increase of the long isoforms of some miRNAs reflects increased 2'-O-methylated miRNA isoforms with age. The increase in 2'-O-methylated miRNA isoforms reflected increased Ago2-loading, but not Ago1-loading of specific miRNA isoforms with age. This raised a question on whether there is global shift in small RNA loading pattern between Ago1 and Ago2 with age. To investigate change in small RNA loading pattern between Ago1 and Ago2 with age, we performed small RNA deep-sequencing of Ago1 vs Ago2-IP small RNAs at 3d and 30d in Drosophila. This analysis revealed global increase of miRNA loading into Ago2, but not into Ago1 with age. 3d and 30d FLAG-HA-Ago2 male flies were collected. Ago1 and Ago2 were immunoprecipitated by anti-Ago1 and anti-FLAG M2 beads respectively. RNA was purified from the beads, P32-labeled, and small RNA fraction was gel-purififed. Small RNA libraries were prepared using Illumina's TruSeq small RNA sample preparation kit (#RS-200-0012, Illumina, Inc. San Diego, CA), following the manufacturer's protocol. The libraries were multiplexed and sequenced on HiSeq2000 platform (Illumina).
Project description:Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide, underscoring the need to improve diagnostic strategies. Platelets play a major role, not only in the process of acute thrombosis during plaque rupture, but also in the formation of atherosclerosis itself. MicroRNAs are endogenous small non-coding RNAs that control gene expression and are expressed in a tissue and disease-specific manner. Therefore they have been proposed to be useful biomarkers. It remains unknown whether differences in miRNA expression levels in platelets can be found between patients with premature CAD and healthy controls. Methodology/Principal Findings In this case-control study we measured relative expression levels of platelet miRNAs using microarrays from 12 patients with premature CAD and 12 age- and sex-matched healthy controls. Six platelet microRNAs were significantly upregulated (miR340*, miR451, miR454*, miR545:9.1. miR615-5p and miR624*) and one miRNA (miR1280) was significantly downregulated in patients with CAD as compared to healthy controls. To validate these results, we measured the expression levels of these candidate miRNAs by qRT-PCR in platelets of individuals from two independent cohorts; validation cohort I consisted of 40 patients with premature CAD and 40 healthy controls and validation cohort II consisted of 27 patients with artery disease and 40 healthy relatives. MiR340* and miR624* were confirmed to be upregulated in patients with CAD as compared to healthy controls in both validation cohorts. Conclusion/Significance Two miRNAs in platelets are significantly upregulated in patients with CAD as compared to healthy controls. miRNA array analysis of isolated platelets from subjects with premature coronary artery disease compared to healthy control subjects.
Project description:Schmitz2014 - RNA triplex formation
The model is parameterized using the
parameters for gene CCDC3 from Supplementary Table S1. The two
miRNAs which form the triplex together with CCDC3 are miR-551b and
This model is described in the article:
Cooperative gene regulation
by microRNA pairs and their identification using a
Schmitz U, Lai X, Winter F,
Wolkenhauer O, Vera J, Gupta SK.
Nucleic Acids Res. 2014 Jul; 42(12):
MicroRNAs (miRNAs) are an integral part of gene regulation
at the post-transcriptional level. Recently, it has been shown
that pairs of miRNAs can repress the translation of a target
mRNA in a cooperative manner, which leads to an enhanced
effectiveness and specificity in target repression. However, it
remains unclear which miRNA pairs can synergize and which genes
are target of cooperative miRNA regulation. In this paper, we
present a computational workflow for the prediction and
analysis of cooperating miRNAs and their mutual target genes,
which we refer to as RNA triplexes. The workflow integrates
methods of miRNA target prediction; triplex structure analysis;
molecular dynamics simulations and mathematical modeling for a
reliable prediction of functional RNA triplexes and target
repression efficiency. In a case study we analyzed the human
genome and identified several thousand targets of cooperative
gene regulation. Our results suggest that miRNA cooperativity
is a frequent mechanism for an enhanced target repression by
pairs of miRNAs facilitating distinctive and fine-tuned target
gene expression patterns. Human RNA triplexes predicted and
characterized in this study are organized in a web resource at
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Project description:The molecular events promoting the initiation of gastric cancer (GC), the third most lethal cancer worldwide, are ill-defined. Here, we demonstrate that upregulation of miR-200 family members is associated with tumor initiation in the gp130F/F mouse model of early GC. The onset of gastric inflammation-associated adenomatous hyperplasia in gp130F/F mice occurs at ~6 weeks of age, with established intestinal-type tumors (3 months of age) progressively growing until a maximum size is reached at 6 months of age, with some evidence of carcinoma in situ. Since this phenotype histologically mimics early stage human GC, we therefore utilized gp130F/F mice as a model to identify miRNAs involved in the initiating molecular events leading to early stage GC. For this purpose, we performed miRNA microarrays on mouse gastric antrum tissue from gp130F/F and sex-matched littermate wild-type (gp130+/+) control mice aged 4 weeks; this age was chosen since antrum tissue from 4 week old (wo) gp130F/F mice is devoid of any histological signs of inflammation or hyperplasia, and is thus comparable to wild-type gastric antrum tissue Overall design: Total RNA from 4 week old mouse antrum samples was subjected to an Affymetrix miRNA 2.0 array which included 722 mouse mature miRNAs and 690 mouse pre-miRNAs (Affymetrix).
Project description:A defining feature of the mammalian liver is polyploidy, a numerical change in the entire complement of chromosomes. The first step of polyploidization involves cell division with failed cytokinesis. Although polyploidy is common, affecting ~90% of hepatocytes in mice and 50% in humans, the specialized role played by polyploid cells in liver homeostasis and disease remains poorly understood. The goal of this study was to identify novel signals that regulate polyploidization, and we focused on microRNAs (miRNAs). First, to test whether miRNAs could regulate hepatic polyploidy we examined livers from Dicer1 liver-specific knockout mice, which are devoid of mature miRNAs. Loss of miRNAs resulted in a 3-fold reduction in binucleate hepatocytes, indicating that miRNAs regulate polyploidization. Secondly, we surveyed age-dependent expression of miRNAs in wild-type mice and identified a subset of miRNAs, including miR-122, that is differentially expressed at 2-3 weeks, a period when extensive polyploidization occurs. Next, we examined Mir122 knockout mice and observed profound, life-long depletion of polyploid hepatocytes, proving that miR-122 is required for complete hepatic polyploidization. Moreover, the polyploidy defect in Mir122 knockout mice was ameliorated by adenovirus-mediated over-expression of miR-122, underscoring the critical role miR-122 plays in polyploidization. Finally, we identified direct targets of miR-122 (Cux1, Rhoa, Iqgap1, Mapre1, Nedd4l and Slc25a34) that regulate cytokinesis. Inhibition of each target induced cytokinesis failure and promoted hepatic binucleation. Conclusion: Our data demonstrate that miR-122 is both necessary and sufficient in liver polyploidization. Among the different signals that have been associated with hepatic polyploidy, miR-122 is the first liver-specific signal identified. These studies will serve as the foundation for future work investigating miR-122 in liver maturation, homeostasis and disease. Livers from C57Bl/6 mice were isolated at defined ages: embryonic day 15.5 (n=3; mixed gender), 2 weeks (n=3; male), 3 weeks (n=3, male) and 7 weeks (n=3; male). Differential miRNA expression was assessed using the nCounter Mouse miRNA Expression Assay Kit (nanoString).
Project description:MicroRNAs (miRNAs) play a important part in post-transcriptional gene regulation and have been shown to control many genes involved in various biological and metabolic processes. There have been extensive studies to discover miRNAs and analyze their functions in model plant species, such as Arabidopsis and rice and other plants. However, the number of miRNAs discovered in grape is relatively low and little is known about miRNAs responded gibberellin during fruit germination. In this study, a small RNA library from gibberellin grape fruits was sequenced by the high throughput sequencing technology. A total of 16,033,273 reads were obtained. 812,099 total reads representing 1726 unique sRNAs matched to known grape miRNAs. Further analysis confirmed a total of 149 conserved grapevine miRNA (Vv-miRNA) belonging to 27 Vv-miRNA families were validated, and 74 novel potential grapevine-specific miRNAs and 23 corresponding novel miRNAs* were discovered. Twenty-seven (36.5%) of the novel miRNAs exhibited differential QRT-PCR expression profiles in different development gibberellin-treated grapevine berries that could further confirm their existence in grapevine. QRT-PCR analysis on transcript abundance of 27 conserved miRNA family and the new candidate miRNAs revealed that most of them were differentially regulated by the gibberellin, with most conserved miRNA family and 26 miRNAs being specifically induced by gibberellin exposure. All novel sequences had not been earlier described in other plant species. In addition, 117 target genes for 29 novel miRNAs were successfully predicted. Our results indicated that miRNA-mediated gene expression regulation is present in gibberellin-treated grape berries. This study led to the confirmation of 101 known miRNAs and the discovery of 74 novel miRNAs in grapevine. Identification of miRNAs resulted in significant enrichment of the gibberellin of grapevine miRNAs and provided insights into miRNA regulation of genes expressed in grape berries. GSM604831 is the control for the gibberellin-treated sample. The mixture samples of young berries (one week after flowering) large berries (five week after flowering after flowering), and old berries (nine week after flowering) treated with gibberellin, respectively, were generated by deep sequencing, in triplicate, using Illumina 1G Genome Analyzer.
Project description:microRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression by targeting specific mRNAs. Altered expression of circulating miRNAs have been associated with age-related diseases including cancer and cardiovascular disease. Although we and others have found an age-dependent decrease in miRNA expression in peripheral blood mononuclear cells (PBMCs), little is known about the role of circulating miRNAs in human aging. Here, we examined miRNA expression in human serum from young (mean age 30 years) and old (mean age 64 years) individuals using next generation sequencing technology and real-time quantitative PCR. Of the miRNAs that we found to be present in serum, three were significantly decreased in 20 older individuals compared to 20 younger individuals: miR-151a-5p, miR-181a-5p and miR-1248. Consistent with our data in humans, these miRNAs are also present at lower levels in the serum of elderly rhesus monkeys. In humans, miR-1248 was found to regulate the expression of mRNAs involved in inflammatory pathways and miR-181a was found to correlate negatively with the pro-inflammatory cytokines IL-6 and TNFa and to correlate positively with the anti-inflammatory cytokines TGFb and IL-10. These results suggest that circulating miRNAs may be a biological marker of aging and could also be important for regulating longevity. Identification of stable miRNA biomarkers in serum could have great potential as a noninvasive diagnostic tool as well as enhance our understanding of physiological changes that occur with age. Examination of microRNAs isolated from human serum from 11 young (mean age 30 yrs) and 11 old (mean age 64 yrs) individuals and from peripheral blood mononuclear cells from one young (30 yr) and one old (64 yr) individual.