MicroRNA (miRNA) expression from human 293 cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE16442 | GEO | 2009/10/01
SECONDARY ACCESSION(S): PRJNA116343
REPOSITORIES: GEO
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Project description:Expression phospho-mimic TRBP enhanced in vitro miRNA production and cellular expression of ectopic miRNA. We used miRNA microarray experiments to determine the effect of expression of phopsho-mimic TRBP on global endogenous miRNA expression. We established isogenic cell lines stably expressing phospho-mutant and phospho-mimic TRBP using Flp-In 293 cells and a Flp Recombinase Target system. After monoclonal cell colonies were selected, cells were cultured for two weeks. Total RNA was harvested when cells were ~75% confluent. Initial analysis was performed using one sample from each experimental group. Subsequent analysis included an additional two samples for each group. miRNA with values less than 500 were not included in analysis.
2010-05-22 | E-GEOD-16442 | biostudies-arrayexpress
Project description:TRBP has two known functions as Dicer co-factor and PKR inhibitor. However, the role of TRBP in miRNA biogenesis is controversial and its regulation of PKR in mitosis remains unexplored. Here, we generate TRBP KO HeLa cells and find that TRBP depletion alters Dicer processing sites of a subset of miRNAs, but does not affect Dicer stability, miRNA abundance, or Argonaute loading. By generating PACT, another Dicer interactor, and TRBP/PACT double-KO cells, we further show that TRBP and PACT do not functionally compensate each other and that only TRBP contributes to Dicer processing. We also report that TRBP is hyperphosphorylated by JNK in M phase when PKR is activated by cellular dsRNAs. Hyperphosphorylation potentiates the inhibitory activity of TRBP on PKR, suppressing PKR in M-G1 transition. By generating the first human TRBP KO, our study clarifies the role of TRBP and unveils negative feedback regulation of PKR through TRBP phosphorylation.
2014-09-16 | GSE61458 | GEO
Project description:TRBP has two known functions as Dicer co-factor and PKR inhibitor. However, the role of TRBP in miRNA biogenesis is controversial and its regulation of PKR in mitosis remains unexplored. Here, we generate TRBP KO HeLa cells and find that TRBP depletion alters Dicer processing sites of a subset of miRNAs, but does not affect Dicer stability, miRNA abundance, or Argonaute loading. By generating PACT, another Dicer interactor, and TRBP/PACT double-KO cells, we further show that TRBP and PACT do not functionally compensate each other and that only TRBP contributes to Dicer processing. We also report that TRBP is hyperphosphorylated by JNK in M phase when PKR is activated by cellular dsRNAs. Hyperphosphorylation potentiates the inhibitory activity of TRBP on PKR, suppressing PKR in M-G1 transition. By generating the first human TRBP KO, our study clarifies the role of TRBP and unveils negative feedback regulation of PKR through TRBP phosphorylation. small RNAs of wild type, TRBP knockout, PACT knockout and TRBP/PACT double knockout cells were sequenced by Illumina Miseq.
2014-09-16 | E-GEOD-61458 | biostudies-arrayexpress
Project description:We generated the cardiac-specific knockout of Trbp (Trbp-cKO) in mice. We profiled the transcriptome in both wild-type and Trbp-cKO hearts, and found numerous genes were deregulated by deletion of Trbp. We also profiled miRNA expression both wild-type and Trbp-cKO hearts, and found expression of a subset of miRNA species was altered in Trbp-cKO hearts.
2015-04-27 | GSE67658 | GEO
Project description:We generated the cardiac-specific knockout of Trbp (Trbp-cKO) in mice. We profiled the transcriptome in both wild-type and Trbp-cKO hearts, and found numerous genes were deregulated by deletion of Trbp. We also profiled miRNA expression both wild-type and Trbp-cKO hearts, and found expression of a subset of miRNA species was altered in Trbp-cKO hearts. Examine expression of mRNAs and miRNAs in wild-type and Trbp-cKO hearts
2015-04-27 | E-GEOD-67658 | biostudies-arrayexpress
Project description:We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr co-factor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its co-factors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs versus passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells.
2014-04-17 | GSE56836 | GEO
Project description:Primary human M1 and M2 macrophages were transfected with different human herpesvirus-derived viral miRNA and the impact on the cellular microRNAs was profiled using microarray. Viral miRNA-mediated impact was assessed on the host cellular microRNA profiles by miRNA microarray analysis. Five different viral miRNA representing 4 different human herpesviruses were overexpressed in M1 and M2 macrophages and the changes in cellular miRNA compared to control mimic were examined. Viral miRNA exhibit unique impact on M1 and M2 miRNA profiles.
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Project description:Mutations in SOD1 (Superoxide Dismutase 1) gene are associated with amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. By employing ascorbate peroxidase (APEX)-based proximity labeling, coupled with LC-MS/MS analysis, we uncovered 37 and 28 proteins exhibiting higher abundance in the proximity proteomes of SOD1G85R and SOD1G93A, respectively, than that of the wild-type SOD1. Immunoprecipitation followed by western blot analysis confirmed the preferential binding of one of these proteins, exportin 5 (XPO5), toward the two mutants of SOD1 over its wild-type counterpart. In line with the established function of XPO5 in pre-miRNA transport, we observed diminished nucleocytoplasmic transport of pre-miRNAs in cells with ectopic expression of the two SOD1 mutants over those expressing the wild-type counterpart. On the other hand, RT-qPCR results revealed significant elevations in mature miRNA in cells expressing the two SOD1 mutants, which are attributed to diminished inhibitory effect of XPO5 on Dicer-mediated cleavage of pre-miRNA to mature miRNA. Together, our chemoproteomic approach led to the revelation of a novel mechanism through which ALS-associated mutants of SOD1 perturb miRNA biogenesis, i.e., through aberrant binding toward XPO5.
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Project description:Triple negative breast cancer (TNBC) is histologically characterized by the absence of the hormone receptors estrogen and progesterone, in addition to having a negative immunostain for HER-2. The aggressiveness of this disease and lack of targeted therapeutic options for treatment is of high clinical importance. MicroRNAs are short 21- to 23 nucleotide endogenous non-coding RNAs that regulate gene expression by binding to mRNA transcripts, resulting in either decreased protein translation or mRNA degradation. Dysregulated expression of miRNAs is now a hallmark of many human cancers. In order to identify a miRNA/mRNA interaction that is biologically relevant to the triple negative breast cancer genotype/phenotype, we initially conducted a miRNA profiling experiment to detect differentially expressed miRNAs in cell line models representing the triple negative (MDA-MB-231), ER+ (MCF7), and HER-2 overexpressed (SK-BR-3) histotypes. We identified human miR-34a expression as being >3-fold down (from its median expression value across all cell lines) in MDA-MB-231 cells, and identified AXL as a putative mRNA target using multiple miRNA/target prediction algorithms. The miR-34a/AXL interaction was functionally characterized through ectopic overexpression experiments with a miR-34a mimic. In reporter assays, miR-34a binds to the putative target site within the AXL 3’UTR to affect luciferase expression. We also observed degradation of AXL mRNA and decreased AXL protein levels, as well as cell signaling effects on AKT phosphorylation and phenotypic effects on cell migration. Finally, we present an inverse correlative trend in miR-34a and AXL expression for both cell line and patient tumor samples.
2010-12-31 | GSE21719 | GEO
Project description:We have used genome editing to generate inactivating deletion mutations in all three copies of the dicer (hdcr) gene present in the human cell line 293T. As previously shown in murine ES cells lacking Dicer function, hDcr-deficient 293T cells are severely impaired for the production of mature microRNAs (miRNAs). Nevertheless, RNA-induced silencing complexes (RISCs) present in these hDcr-deficient cells are readily programmed by transfected, synthetic miRNA duplexes to repress mRNAs bearing either fully or partially complementary targets, including targets bearing incomplete seed homology to the introduced miRNA. Using these hDcr-deficient 293T cells, we demonstrate that human pre-miRNA processing can be effectively rescued by ectopic expression of the Drosophila Dicer 1 protein, but only in the presence of the PB isoform of Loquacious (Loqs-PB), the fly homolog of the hDcr co-factor TRBP. In contrast, Drosophila Dicer 2, even in the presence of its co-factors Loqs-PD and R2D2, was unable to support human pre-miRNA processing. Interestingly, although ectopic Drosophila Dicer 1/Loqs-PB or hDcr both rescued pre-miRNA processing effectively in these hDcr-deficient cells, there were significant differences in the ratio of the miRNA isoforms that were produced, especially in the case of miR-30 family members, and we also noted differences in the relative expression level of miRNAs versus passenger strands for a subset of human miRNAs. These data demonstrate that the mechanisms underlying the accurate processing of pre-miRNAs are largely, but not entirely, conserved between mammalian and insect cells. Series includes three datasets of total small RNA reads from wild type and Dicer negative 293T cells. Also included are total small RNA reads of the Dicer-negative cell line NoDice(4-25) transfected with a vector expressing human Dicer, Drosophila Dicer1, or mock transfected. RISC-associated small RNAs identified by ribonucleoprotein immunoprecipitation (RIP-Seq) in wild type 293T, Dicer-negative NoDice(4-25) line, and NoDice(4-25) transfected with either hsa-miR-155 or kshv-miR-K12-11 miRNA duplexes. The final data series are PAR-CLIP libraries which identified microRNA targets in untransfected NoDice(4-25) cells and NoDice(4-25) cells transfected with either hsa-miR-92a, hsa-miR-155, kshv-miR-K12-11 miRNA duplexes
2014-04-17 | E-GEOD-56836 | biostudies-arrayexpress