ABSTRACT: We examined hypoxia responsing miRNAs in HUVEC and MA148cells. Microarray studies revealed hypoxia can change microRNA expression in HUVEC and MA148 cells. 8 HUVEC samples (4 controls and 4 experimentals) and 4 MA148 samples (2 controls and 2 experimentals)
Project description:The high rates of mortality associated with epithelial ovarian cancer (EOC) are a direct consequence of its metastatic nature. Metastasis is dependent on many factors, among which activation of angiogenesis is most significant. Angiogenesis is, in turn, contingent upon the cellular response to hypoxia within the tumor microenvironment. Hypoxia-inducible factor 1 (HIF1) is a transcription factor composed of HIF1alpha and HIF1beta subunits and is the master regulator of the hypoxic response. It is therefore a critical mediator of tumor angiogenesis and metastasis. Regulation of HIF1 is primarily at the level of protein. In normoxia, the HIF1alpha subunit is hydroxylated via an oxygen- and iron-dependent mechanism and targeted for destruction. In hypoxia, low oxygen levels preclude hydroxylation and HIF1alpha is stabilized, allowing for its association with constitutively expressed HIF1beta to form bioactive HIF1. We have identified a novel mechanism of HIF1alpha regulation in EOC cells that involves microRNAs (miRs), ~22 nucleotide, non-coding RNA molecules that repress translation of target mRNAs by binding their 3’ untranslated regions (UTRs). Using microarray and qPCR analysis, we found that levels of miR-199a-1, a miR that is predicted in silico to target the HIF1alpha 3’ UTR, were reduced under hypoxia in EOC cells. We further demonstrated that miR-199a-1 directly targets the HIF1alpha 3’ UTR and overexpression of miR-199a-1 suppresses HIF1alpha protein levels and HIF1-driven gene expression. Moreover, cells stably overexpressing miR-199a-1 exhibit marked defects in migratory ability. These data were corroborated by our in vivo findings, which demonstrated that overexpression of miR-199a-1 causes significant reductions in tumor vessel density and tumor burden in nude mice. These findings provide insight into non-canonical, miR- and iron-based mechanisms of HIF1 regulation that may have important implications in the progression of EOC. miRNA expression analysis of A2780 epithelial ovarian cancer cells by microarrays
Project description:Our analyses of microRNA expression profiles involve the hybridization of fluorescently labeled miRNA samples to custom made, DNA microarrays based on the GAPSII coated slides. We hybridized different amounts of microRNAs from the mirVana miRNA Reference Panel v9.1 to microarrays. Keywords: Expression Three different concentrations of miRNAs from the commercial standrads were labelled and hybridized to three microarrays.
Project description:Ago2 binds mature microRNAs (miRNAs) to regulate gene expression. A mutant Ago2 is constructed to render it unable to bind miRNAs and inhibit mRNA translation. Human 293T cells were transiently transfected with a plasmid that encoded a FLAG-tagged wildtype human Ago2 or mutant Ago2. FLAG IP was preformed, and assoicated RNA was isolated and subjected miRNA microarray analysis.
Project description:Endotoxin/LPS tolerance is a tightly regulated phenomenon, which, during infection, prevents systemic hyper-inflammation. Here we report for the first time that morphine reversal of endotoxin tolerance resulting in persistent inflammation thus contributing to septicemia and septic shock. We further report that this regulation is mediated by LPS-induced down-regulation of microRNAs 146a and 155. However, only over-expression of miR-146a, but not miR-155 abrogates morphine mediated hyper-inflammation, while antagonizing miR-146a (but not miR-155) augments morphine mediated hyper-inflammation. Hence, miR-146a could be the potential therapeutic target for morphine-mediated abrogation of endotoxin tolerance. All treatments done in vivo. Morphine implanted subcuteniously, LPS administered as intraperitoneal injection.
Project description:Our genome wide analyses of microRNA expression profiles involve the hybridization of fluorescently labeled RNA samples to custom made, DNA microarrays based on the GAPSII coated slides. We describe a simple and effective method to regenerate such custom microarrays. Our protocol entails the use of a very low concentration of sodium hydroxide in a low salt buffer to strip RNA molecules from the arrays. The solution is also capable of removing DNA molecules hybridized to the slides, while preserving the slide coating and printed DNA probes. Slides can be stripped and reused at least twice without significantly sacrificing data quality. Keywords: expression study, new vs. stripped array comparison There are two stripping conditions in this study (1mM NaOH and 2mM NaOH in SSC buffer). For 1mM NaOH treatment, new, once-, twice-, and triple-stripped arrays are studies. For 2mM NaOH treatment, new and once-stripped arrays are measured.
Project description:Our genome wide analyses of microRNA expression profiles involve the hybridization of fluorescently labeled RNA samples to custom made, DNA microarrays based on the GAPSII coated slides. We describe the differences in genome wide miRNA expressions between wild type 293 T mammalian cells and Ago2 transformed stable cell lines. Keywords: Expression Comparing one control and three Ago2-overexpressing stable cell lines (A, B, C). One replicate per array.
Project description:We examined the differential expression of miRNAs in vinyl carbamate (VC)-induced lung tumors in A/J mice and assessed if the chemopreventive agent indole-3-carbinol (I3C) reversed the effects of the carcinogen. Microarray studies revealed some up-regulated microRNAs, but down-regulation of several microRNAs in carcinogen-treated mice relative to vehicle-treated mice. Keywords: Expression micro and control RNA from control treated (3 replicates), vinyl carbamate treated (4 replicates) and vinyl carbamate and indole-3-carbinol treated mice was hybridized versus synthetic human DNA to a custom multi-organism oligonucleotide array.
Project description:To study the relationship between microRNAs and μ-opioid receptor (MOR) signaling, we examined microRNA expression after chronic morphine or fentanyl treatment in rat primary hippocampal neurons and in mouse hippocampus. Mouse cerebellum region was also tested as a negative control to eliminate microRNA expression changes unrelated to MOR signaling, as the cerebellum is essentially devoid of MOR. We identified a number of microRNAs that altered their expression upon treatment with both morphine and fentanyl in the rat and mouse systems. There were, however, some microRNAs that changed in response to morphine, or fentanyl, but not both. Keywords: Expression profiling There are up to three biological replicates (indicated by 1, 2, and 3) of primary hippocampal neurons from new born rats and the cerebellum and hippocampus regions from adult mice treated for three days (control, morphine, and fentanyl). The biological replicates were from experiments performed on different dates. Each biological replicate contained cells or tissues collected from multiple animals so that enough RNA could be extracted for RNA analysis. RNA was labelled with a green dye, mixed with a reference DNA sample labelled with a red dye. The reference DNA contained a number of synthetic DNA oligos with mature microRNA sequences that served to verify microarray hybridization. RNA signals were in ch1, DNA signals ch2.
Project description:Liver is one of the few organs that have the capacity to regenerate in response to injury. We carried out genome-wide miRNA microarray studies during liver regeneration in rats after 70% partial hepatectomy (PH) at early and mid-time points to more thoroughly understand their role. At 3, 12 and 18 hrs post-PH ~ 40% of the miRNAs tested were up-regulated. Conversely, at 24 hrs post-PH, ~ 70% of miRNAs were down-regulated. Further, we established that the genome-wide down-regulation of miRNA expression at 24 hrs was also correlated with decreased expression of genes such as Rnasen, Dgcr8, Dicer, Tarbp2 and Prkra that are associated with miRNA biogenesis. To determine if a potential negative feedback loop between miRNAs and their regulatory genes existed, 11 candidate miRNAs which were predicted to target the above genes were examined and found to be up-regulated at 3 hrs post-PH. Using reporter and functional assays, we determined that expression of these miRNA-processing genes could be regulated by a subset of miRNAs and some miRNAs could target multiple miRNA biogenesis genes simultaneously. We also demonstrated that over-expression of these miRNAs inhibited cell proliferation and modulated the cell cycle in both Huh-7 human hepatoma cells and primary rat hepatocytes. From these observations, we postulated that selective up-regulation of miRNAs in the early-phase after PH was involved in the priming and commitment to liver regeneration, while the subsequent genome-wide down-regulation of miRNAs was required for efficient recovery of liver cell mass. Conclusion: Our data suggest that miRNA changes are regulated by negative feedback loops between miRNAs and their regulatory genes that may play an important role in the steady-state regulation of liver regeneration. Sham and Partial Hepatectomized livers at various time points.