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A Daphnane Diterpenoid Isolated from Wikstroemia polyantha induces an inflammatory response and modulates miRNA activity

ABSTRACT: MicroRNAs (miRNAs) are endogenously expressed single-stranded ~21–23 nucleotide RNAs that inhibit gene expression post-transcriptionally by binding imperfectly to elements usually within the 3′untranslated region (3′UTR) of mRNAs. Small interfering RNAs (siRNAs) mediate site-specific cleavage by binding with perfect complementarity to RNA. Here, a cell-based miRNA reporter system was developed to screen for compounds from marine and plant extracts that inhibit miRNA or siRNA activity. The daphnane diterpenoid genkwanine M (GENK) isolated from the plant Wikstroemia polyantha induces an early inflammatory response and can moderately inhibit miR-122 activity in the liver Huh-7 cell line. GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells. In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity.

ORGANISM(S): Homo sapiens

PROVIDER: GSE39002 | GEO | 2012/06/29

SECONDARY ACCESSION(S): PRJNA169563

REPOSITORIES: GEO

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A Daphnane Diterpenoid Isolated from Wikstroemia polyantha induces an inflammatory response and modulates miRNA activity

Project description:MicroRNAs (miRNAs) are endogenously expressed single-stranded ~21M-bM-^@M-^S23 nucleotide RNAs that inhibit gene expression post-transcriptionally by binding imperfectly to elements usually within the 3M-bM-^@M-2untranslated region (3M-bM-^@M-2UTR) of mRNAs. Small interfering RNAs (siRNAs) mediate site-specific cleavage by binding with perfect complementarity to RNA. Here, a cell-based miRNA reporter system was developed to screen for compounds from marine and plant extracts that inhibit miRNA or siRNA activity. The daphnane diterpenoid genkwanine M (GENK) isolated from the plant Wikstroemia polyantha induces an early inflammatory response and can moderately inhibit miR-122 activity in the liver Huh-7 cell line. GENK does not alter miR-122 levels nor does it directly inhibit siRNA activity in an in vitro cleavage assay. Finally, we demonstrate that GENK can inhibit HCV infection in Huh-7 cells. In summary, the development of the cell-based miRNA sensor system should prove useful in identifying compounds that affect miRNA/siRNA activity. Control (no drug treatment): 1 hour and 4 hour samples, 3 replicates each; Drug-treated: 1 hour and 4 hours samples, 3 replicates each

2012-06-28 | E-GEOD-39002 | biostudies-arrayexpress

Hepatic ARID3A facilitates liver cancer malignancy by cooperating with CEP131 to regulate an embryonic stem cell-like gene signature

Project description:Huh 7 cells were infected with lentivirus or transfected with siRNA using Lipofectamine RNAiMAX to overexpress or knockdown indicated genes. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) was performed using indicated antibodies to study the H3K9me2 status after certain treatment in Huh7 cells. Cleavage Under Targets and Tagmentation (CUT&Tag) was performed to explore the chromatin binding sites of ARID3A. Whole-transcriptome sequencing (RNA-Seq) was performed to identify the downstream target genes regulated by ARID3A or KDM3A in Huh 7 cells.

2022-08-10 | GSE193726 | GEO

Functional analysis of Arabidopsis ARGONAUTE1 using a slicer-defective mutant: Small RNA immunoprecipitation

2012-09-28 | GSE40258 | GEO

Functional analysis of Arabidopsis ARGONAUTE1 using a slicer-defective mutant: Transcript immunoprecipitation

2012-09-28 | GSE40256 | GEO

Functional analysis of Arabidopsis ARGONAUTE1 using a slicer-defective mutant: Small RNA immunoprecipitation

Project description:The Arabidopsis ARGONAUTE (AGO) protein AGO1 associates with microRNA (miRNA) and specific classes of short-interfering RNA (siRNA). AGO1-small RNA complexes recognize target RNA transcripts through base-pairing interactions and inhibit translation of target RNAs through endonucleolytic cleavage (slicing) or non-degradative mechanisms. The PIWI domain of AGO1 contains a metal-coordinating triad [Asp-Asp-His] (DDH) that is required for slicer activity. Here, we compared the activities of wild type (DDH) and slicer active-site defective (DAH) forms of AGO1 by sequencing small RNA and target transcript RNAs that co-immunoprecipitated with hemagglutinin (HA)-tagged AGO1 proteins. We found that the population of miRNA that associated with both AGO1-DDH and AGO1-DAH proteins largely overlapped, suggesting that cleavage activity does not affect miRNA maturation. In contrast, slicer-defective AGO1-miRNA complexes associated with target RNA more effectively than did wild type AGO1-miRNA. These data indicate that slicer-defective AGO proteins can be used as an approach to capture AGO-small RNA-target RNA ternary complexes more efficiently for genome-wide analyses. AGO1-DDH (wild type) AGO1-DAH (slicer mutant) proteins were immunoprecipitated (N-terminal 3xHA) from Arabidopsis (Columbia) flower (stages 1-12) lysate. Immunoprecipitations were also done from control plants transformed with vector only. Small RNA from immunoprecipitate fractions of vector, AGO1-DDH and AGO1-DAH were sequenced.

2012-09-28 | E-GEOD-40258 | biostudies-arrayexpress

Effect of miR-122 overexpression of mRNA distribution on the polyribosomes

Project description:The aim of this study was to identify at genome-wide level, the effect of miR-122 on the distribution of its responsive genes on the polyribosomes. For this purpose human hepatoma cells Huh-7 were transfected for 48 hrs with LNA-based miR-122 inhibitors (122-PM) or miR-122 mimics (122-MIM). Following the treatment with cycloheximide, cells were lysed and layered on a 10-50% sucrose gradient. Following ultracetrifigation the polyribosomes were separated in 12 different fractions

2023-06-14 | GSE234690 | GEO

Functional analysis of Arabidopsis ARGONAUTE1 using a slicer-defective mutant: Transcript immunoprecipitation

Project description:The Arabidopsis ARGONAUTE (AGO) protein AGO1 associates with microRNA (miRNA) and specific classes of short-interfering RNA (siRNA). AGO1-small RNA complexes recognize target RNA transcripts through base-pairing interactions and inhibit translation of target RNAs through endonucleolytic cleavage (slicing) or non-degradative mechanisms. The PIWI domain of AGO1 contains a metal-coordinating triad [Asp-Asp-His] (DDH) that is required for slicer activity. Here, we compared the activities of wild type (DDH) and slicer active-site defective (DAH) forms of AGO1 by sequencing small RNA and target transcript RNAs that co-immunoprecipitated with hemagglutinin (HA)-tagged AGO1 proteins. We found that the population of miRNA that associated with both AGO1-DDH and AGO1-DAH proteins largely overlapped, suggesting that cleavage activity does not affect miRNA maturation. In contrast, slicer-defective AGO1-miRNA complexes associated with target RNA more effectively than did wild type AGO1-miRNA. These data indicate that slicer-defective AGO proteins can be used as an approach to capture AGO-small RNA-target RNA ternary complexes more efficiently for genome-wide analyses. AGO1-DDH (wild type) AGO1-DAH (slicer mutant) proteins were immunoprecipitated (N-terminal 3xHA) from Arabidopsis (Columbia) flower (stages 1-12) lysate. Immunoprecipitate fractions were treated with nuclease P1 before cleanup to trim free RNA ends, and therefore enrich samples in AGO1 target sites. All samples were treated with Ribominus (Life Technologies) to reduce ribosomal RNA abundance. Transcript fragments from two replicates of AGO1-DDH and AGO1-DAH immunoprecipitates were sequenced. Ribominus-treated total RNA (input controls) was also sequenced for each replicate.

2012-09-28 | E-GEOD-40256 | biostudies-arrayexpress

miR-122-dependent binding of TNRC6 to HCV RNA

Project description:The liver-specific microRNA, miR-122, is an essential host factor for replication of hepatitis C virus (HCV), an important infectious cause of chronic liver disease and hepatocellular carcinoma. miR-122 stabilizes the positive-strand HCV RNA genome and promotes viral RNA synthesis by binding two closely spaced sites (S1 and S2) near the 5’ end of the genome in association with Ago2. Ago2 is essential for both host factor activities, but whether other host proteins are involved is unknown. Using a quantitative proteomics approach, we identified TNRC6A (GW182) and its paralogs (TNRC6B and TNRC6C), as functionally important components of the miR-122/Ago2 host factor complex binding HCV RNA. Depletion of any two TNRC6 proteins reduced HCV replication in Huh-7.5 cells,but did not reduce viral RNA stability or translational activity, but rather dampened miR-122 stimulation of viral RNA synthesis. However, TNRC6 depletion had no effect on replication of HCV in which S2 was mutated so that miR-122 binds only S1, whereas it significantly enhanced replication when S1 was mutated and only S2 bound by miR-122. Consistent with this, we found that TNRC6 proteins preferentially associate with the S1 site, and that the association of Ago2 with S2 is increased in TNRC6-depleted cells. Collectively, these data suggest a model in which TNRC6 proteins, which are known to interact with Ago2, preferentially direct the miR-122/Ago2 complex to S1 while restricting its association with S2, thereby fine tuning the spatial organization of miR-122/Ago2 complexes bound to the viral RNA.

2019-04-24 | PXD013054 | Pride

Evaluation of the effect of antimiR-122 administration on the liver, spleen and heart miRNome

Project description:To determine the effect of antimiR-122 administration on the mouse miRNome. To functionally investigate a possible link between miR-122 and iron metabolism we inhibited miR-122 by a single, intraperitoneal injection of Locked Nucleic Acid (LNA)-modified antimiR oligonucleotides into age- and sex-matched C57Bl/6 WT mice. To inhibit miR-122 specifically, we injected an antimiR compound with perfect complementarity to miR-122 [perfect match (PM); PM_antimiR-122]. As negative controls, mice were either injected with an LNA control compound with two mismatches (2MM, 2MM_antimiR-122) or the vehicle control (SAL; 0.9% NaCl). Mice were sacrificed three and six weeks after injection. Independent of treatment, mice were viable and exhibited no overt physical or behavioral abnormalities. To exclude that PM_antimiR-122 administration disturbs the expression of other miRNAs we analyzed miRNA expression profiles in the livers, hearts and spleens of the same mice.

2010-10-20 | E-GEOD-24786 | biostudies-arrayexpress

Transcription profiling of human A549 lung cancer cells treated with compounds from a series of inhibitors developed against the human LIM kinases LIMK1 and LIMK2

Project description:To investigate an unknown mechanism of cytotoxicity, A549 human lung-cancer cells were treated with compounds from a series of inhibitors developed against the human LIM kinases LIMK1 and LIMK2. Compounds 1 and 2 inhibit LIM kinase activity in vitro and affect cell proliferation and survival in vivo. Compounds 3 and 4 inhibit LIM kinases but do not affect cell survival or proliferation. Compounds 5 and 6 affect proliferation and survival but do not inhibit LIM kinases. Nocodazole was included as a comparator because the compounds were known to affect microtubule stability. A treatment of 7 hours was used to examine events prior to apoptosis, while the dose levels captured both cytotoxicity and inhibition of LIMKs (Compounds 1 and 2), LIMK inhibition alone ( Compounds 3 and 4) or cytotoxicity alone (Compounds 5, 6, and Nocodazole).

2008-09-17 | E-TABM-450 | biostudies-arrayexpress

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