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Identification of miR-21 targets in Jurkat cells by AGO2 immunoprecipitation

ABSTRACT: In order to identify miR-21 targets by a biochemical high-throughput method, we immunopurified RISC Complex and associated mRNAs in both control and miR-21 overexpressing Jurkat cells. Following transduction of miR-21 encoding construct (or a cognate control vector) in Jurkat cells, we used microarray technology to profile Total RNA, AGO2 Immunopurified RNA and control IgG purified RNA.

ORGANISM(S): Homo sapiens

SUBMITTER: Teresa Colombo

PROVIDER: E-GEOD-37212 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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miR-21 is a negative modulator of T-cell activation.

Carissimi Claudia C Carucci Nicoletta N Colombo Teresa T Piconese Silvia S Azzalin Gianluca G Cipolletta Emanuela E Citarella Franca F Barnaba Vincenzo V Macino Giuseppe G Fulci Valerio V

Biochimie 20141007

microRNAs (miRNAs) are a class of small non-coding RNAs acting as post-transcriptional regulators of gene expression and play fundamental roles in regulating immune response and autoimmunity. We show that memory T-lymphocytes express higher levels of miR-21 compared to naïve T-lymphocytes and that miR-21 expression is induced upon TCR engagement of naïve T-cells. We identify bona fide miR-21 targets by direct immuno-purification and profiling of AGO2-associated mRNAs in Jurkat cells over-express ...[more]

PMID: 25304039

Similar Datasets

Identification of miR-21 targets in Jurkat cells by AGO2 immunoprecipitation

Project description:In order to identify miR-21 targets by a biochemical high-throughput method, we immunopurified RISC Complex and associated mRNAs in both control and miR-21 overexpressing Jurkat cells.

2014-10-15 | GSE37212 | GEO

mRNA-seq and AGO2-RIP-seq of JURKAT T-cell acute lymphoblastic leukemia cell line upon overexpression of hsa-miR-143-3p

Project description:The aim of this study was to investigate the effect of hsa-miR-143-3p on global gene expression in T-cell acute lymphoblastic leukemia (T-ALL) and to unravel its direct target genes. For this purpose we selected Jurkat T-ALL cell line characterized by low expression of this miRNA. The cells were transduced either with pCDH-CMV-MCS-EF1_GFP_puro empty vector (System Biosciences) or pCDH-CMV-MCS-EF1_GFP pre-mir-143 vector, each variant in three biological replicates. Total RNA was isolated from each samples. Additionally, RISC complexes containing miRNAs bound to their target mRNAs were isolated by AGO2 immunoprecipitation and the obtained fraction of RISC-bound RNA was isolated from each sample. All 12 libraries (6 total RNA samples and 6 AGO2-RIP samples) were sequenced on Illumina NovaSeq6000 platform, in 2x150 mode, with a coverage of 60M paired reads/sample.

2024-08-31 | E-MTAB-13462 | biostudies-arrayexpress

Experimental identification of apoptosis-associated miR-21 target genes in Jurkat T-cells using a high-throughput experimental approach

Project description:MiR-21 is an important suppressor of T-cell apoptosis that is also widely overexpressed in many types of cancers. The exact mechanisms related to the anti-apoptotic effect of miR-21 is not well understood. In this study, we applied AGO2 RNA Immunoprecipitation followed by gene expression profiling (RIP-Chip) in Jurkat cells to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon Î±CD3/Î±CD28 activation of Jurkat cells. Inhibition of miR-21 resulted in reduced cell growth and induced apoptosis. Upon AGO2-RIP-Chip, we observed an overall increased enrichment of miR-21 target genes in the IP fraction of miR-21-overexpressing Jurkat cells as compared to the IP fraction of empty vector control cells. We noted a systematic decrease in transcript levels of predicted miR-21 target genes compared to EV control. We identified 72 genes that were 2-fold enriched in the AGO2-IP fraction of miR-21-overexpressing cells that contained a predicted miR-21 binding site. Of these, 71 were enriched 2-fold more in the miR-21-overexpressing cells as compared to EV Jurkat cells. The target gene for which the enrichment was most prominently increased upon miR-21 overexpression was the pro-apoptotic protein LATS1. Luciferase reporter assays confirmed direct targeting of the LATS1 3'UTR by miR-21. In line with the luciferase results, Western blot analysis revealed a decrease in LATS1 upon miR-21 inhibition. LATS1 qRT-PCR analysis in primary T-cells showed that LATS1 levels decrease upon T-cell stimulation while the miR-21 levels increase. Collectively, these data identify the miR-21 target LATS1 as a likely candidate whose inhibition contributes to the anti-apoptotic function of miR-21 in T-cells and perhaps also many types of cancers. Gene expression array on Jurkat cells overexpressing miR-21 and empty vector (EV).

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AGO2 bound short RNAs in Jurkat cell line

Project description:Characterization of AGO2 bound short RNAs from nuclear extracts of Jurkat cell line. Nuclei were isolated from cells; a fraction of Nuclear lysates was harvested and sequenced as the input samples. The rest of nuclear lysates were Immunoprecipitated using AGO2 monoclonal antibodies or Isotype matched IgG. Following immunoprecipitation, RNA was extracted from AGO2 or control IgG IP for short RNA sequencing. Two independent biological replicates were carried out.

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Microarray dataset of Jurkat cells following miR-93 over-expression

Project description:miR-93 is often dysregulated in several tumor cell lines, including lymphoblastic leukemias. This dataset can represent a further insight into gene expression changes and GO Terms associated with miR-93 over-expression. miR-93 was over-expressed in Jurkat cells by transduction with a lentiviral transgenic construct encoding for miR-93 under a PGK promoter. A cognate vector encoding for a control hairpin was used to generate control cell line. mRNA Microarray gene expression profiling of Jurkat cells tranduced with either a miR-93 sponge or control construct. 3 biological replicas have been performed.

2016-03-21 | E-MTAB-4588 | biostudies-arrayexpress

Experimental identification of apoptosis-associated miR-21 target genes in Jurkat T-cells using a high-throughput experimental approach

Project description:MiR-21 is an important suppressor of T-cell apoptosis that is also widely overexpressed in many types of cancers. The exact mechanisms related to the anti-apoptotic effect of miR-21 is not well understood. In this study, we applied AGO2 RNA Immunoprecipitation followed by gene expression profiling (RIP-Chip) in Jurkat cells to identify apoptosis-associated miR-21 target genes. We showed that expression of miR-21 rapidly increases upon αCD3/αCD28 activation of Jurkat cells. Inhibition of miR-21 resulted in reduced cell growth and induced apoptosis. Upon AGO2-RIP-Chip, we observed an overall increased enrichment of miR-21 target genes in the IP fraction of miR-21-overexpressing Jurkat cells as compared to the IP fraction of empty vector control cells. We noted a systematic decrease in transcript levels of predicted miR-21 target genes compared to EV control. We identified 72 genes that were 2-fold enriched in the AGO2-IP fraction of miR-21-overexpressing cells that contained a predicted miR-21 binding site. Of these, 71 were enriched 2-fold more in the miR-21-overexpressing cells as compared to EV Jurkat cells. The target gene for which the enrichment was most prominently increased upon miR-21 overexpression was the pro-apoptotic protein LATS1. Luciferase reporter assays confirmed direct targeting of the LATS1 3'UTR by miR-21. In line with the luciferase results, Western blot analysis revealed a decrease in LATS1 upon miR-21 inhibition. LATS1 qRT-PCR analysis in primary T-cells showed that LATS1 levels decrease upon T-cell stimulation while the miR-21 levels increase. Collectively, these data identify the miR-21 target LATS1 as a likely candidate whose inhibition contributes to the anti-apoptotic function of miR-21 in T-cells and perhaps also many types of cancers.

2016-08-03 | GSE85116 | GEO