MiR-17 seed family specific targets in two Hodgkin lymphoma cell lines
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE16053 | GEO | 2009/10/13
SECONDARY ACCESSION(S): PRJNA114997
REPOSITORIES: GEO
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Project description:This SuperSeries is composed of the following subset Series: GSE11079: mRNAs associated with Ago2: Ago2, Ago2+miR-1, Ago2+miR-124, mock transfected GSE11080: Expression data HEK293T cells transfected with either Ago2, Ago2 + miR-1, Ago2 + miR-124, mock GSE11081: miRNA arrays: Ago2 transfected vs. Mock, Ago2 IP vs. total RNA Keywords: SuperSeries Refer to individual Series
2008-04-07 | E-GEOD-11082 | biostudies-arrayexpress
Project description:Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays, and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-Sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs, and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes, and upregulating pluripotency-associated genes such as Lin28. For RNA-IP experiments, cells were transfected with 300 pmol of miR-294 or cel-239b control miRNA (Dharmacon) and harvested 12-16 hr after transfection for immunoprecipitation. Following RNA-immunoprecipitation of Ago2-myc, RNA from the INPUT (total RNA) and IP were subjected to library preparation and sequenced by SOLiD. There are 10 samples from Dicer1-null mouse ES cells, which are essentially 5 sample pairs of INPUT (A) and its corresponding RNA-IP (B): Ago2-myc transfected with miR-294 (sampe 1), 2 replicates of Ago2-myc-MUT (catalytically inactive) transfected with miR-294 (sample 2 & 5), 2 replicates of Ago2-myc transfected with cel-239b (sample 3 & 4).
2010-02-27 | E-GEOD-20199 | biostudies-arrayexpress
Project description:MicroRNAs (miRNAs) are small non-coding RNAs involved in the posttranscriptional regulation of gene expression. Deregulated miRNA levels have been reported in Burkitt lymphoma (BL) compared to normal B cells and a number of miRNA target genes have been identified in BL cell lines. Here, we determined for the first time the miRNA targetomes of primary BL tumors in comparison with normal B cells. AGO2-RNA immunoprecipitation (AGO2-RIP) in two frozen diagnostic BL tissue samples and three CD19+ B-cell samples isolated from routinely removed tonsils revealed distinct miRNA targetomes of BL and normal B cells. Targets of the miR-17~92 cluster were significantly more frequently AGO2-IP enriched in BL while targets of miR-29 and miR-150 were more frequently AGO2-IP enriched in normal B cells. Furthermore, we showed that miRNA target genes in BL are mainly involved in cell cycle and cell death. Immunohistochemistry on BL tumor samples and tonsil tissues confirmed altered protein levels for two out of six selected miRNA targets, in line with the differential AGO2-IP enrichment between BL and normal B cells. A comparison of AGO2-IP enriched genes in primary BL cases with BL cell lines indicated that approximately half of the miRNA targets may be missed in studies using BL cell lines, while almost 60% of miRNA target genes identified in cell lines were not AGO2-IP enriched in primary BL cases. Our results demonstrate both the necessity and feasibility of studying miRNA-target interactions in primary tumors.
2018-01-19 | GSE107325 | GEO
Project description:Micro RNAs (miRNAs) miR-130a, miR-203 and miR-205 are jointly downregulated in prostate cancer and act as repressors of AR-signaling. MiRNAs are small non-coding RNAs that regulate the expression of specific mRNA targets mainly by translational repression, mRNA deadenylation or cleavage. Reconstitution of these lost miRNAs in the LNCaP PCa cell line cause morphology changes, growth arrest, and apoptosis, increasing when the miRNAs were co-expressed. This series identifies direct targets of miR-130a, miR-203, and miR-205 by AGO2-RNA co-immunoprecipitation as described by (Beitzinger et al. 2007) upon miRNA reconstitution in LNCaP cells and analyzing AGO2-bound mRNAs using Affymetrix Genechips. Relative levels of AGO2 bound versus total RNA expression were compared between miRNA reconstituted and miR-scr transfected samples. Three arrays each for AGO2-bound RNA upon reconstitution of miR-130a, miR-203, miR-205, a scramble miRNA, and three arrays each for total RNA upon reconstitution of miR-130a, miR-203, miR-205, a scramble miRNA.
2012-03-08 | E-GEOD-22979 | biostudies-arrayexpress
Project description:Micro RNAs (miRNAs) miR-130a, miR-203 and miR-205 are jointly downregulated in prostate cancer and act as repressors of AR-signaling. MiRNAs are small non-coding RNAs that regulate the expression of specific mRNA targets mainly by translational repression, mRNA deadenylation or cleavage. Reconstitution of these lost miRNAs in the LNCaP PCa cell line cause morphology changes, growth arrest, and apoptosis, increasing when the miRNAs were co-expressed. This series identifies direct targets of miR-130a, miR-203, and miR-205 by AGO2-RNA co-immunoprecipitation as described by (Beitzinger et al. 2007) upon miRNA reconstitution in LNCaP cells and analyzing AGO2-bound mRNAs using Affymetrix Genechips. Relative levels of AGO2 bound versus total RNA expression were compared between miRNA reconstituted and miR-scr transfected samples.
2012-03-08 | GSE22979 | GEO
Project description:miRNA arrays: Ago2 transfected vs. Mock, Ago2 IP vs. total RNA
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Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs. Two replicates of four cDNA libraries were submitted to deep sequencing: an RNA M-bM-^@M-^\InputM-bM-^@M-^] sample from RNA-12-transfected cells; a sample of the Ago2-immunopurified RNA from cells treated with transfection reagent (M-bM-^@M-^\MockM-bM-^@M-^]); a sample of the Ago2-immunopurified RNA from RNA-12-treatment (M-bM-^@M-^\Ago2-IPM-bM-^@M-^]); and the sample from miR-CLIP-purified RNA (M-bM-^@M-^\miR-CLIPM-bM-^@M-^]).
2014-12-22 | E-GEOD-62677 | biostudies-arrayexpress
Project description:Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs. Two replicates of four cDNA libraries were submitted to deep sequencing: an RNA M-bM-^@M-^\InputM-bM-^@M-^] sample from RNA-7-transfected cells; a sample of the Ago2-immunopurified RNA from cells treated with transfection reagent (M-bM-^@M-^\MockM-bM-^@M-^]); a sample of the Ago2-immunopurified RNA from RNA-7-treatment (M-bM-^@M-^\Ago2-IPM-bM-^@M-^]); and the sample from miR-CLIP-purified RNA (M-bM-^@M-^\miR-CLIPM-bM-^@M-^]).
2014-12-22 | E-GEOD-62675 | biostudies-arrayexpress
Project description:Targetome of miR-124/miR-132 was enriched in HEK293T cells, a cell line which lacks the expression of these miRNA. The targetome was captured using miR-CLIP, a transfection-based double-purification crosslinking and immunoprecipitation (CLIP) method. Therefore HEK293T cells were transfected with miR-124/miR-132 miR-CLIP probes, which are trioxsalen, biotin bis-modified pre-miRNAs. Unspecific RNA-protein and trioxsalen mediated RNA-RNA crosslinking were induced by UV-irradiation before cell lysis. Total RNA (input samples) was enriched for AGO2-bound RNA using AGO2 immunoprecipitation, by depleting non-RISC associated RNAs. A second enrichment for specific targets of the transfected miRNA is performed using magnetic streptavidin-beads. This step relies on the interaction of the biotinylated probe, on which the RNA target is crosslinked, with the beads. The method was shown to deliver high-quality miRNA targetomes of both 5p and 3p miRNAs.
2020-11-23 | E-MTAB-8517 | biostudies-arrayexpress
Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP). Ribonucleoprotein IP was performed using the RIP-Assay kit for microRNA (MBL) according to the protocol described by the manufacturer. Briefly, anti-EIF2C2/Ago2 monoclonal antibody (Novus Biologicals, LLC) was incubated with Protein G plus agarose beads (Pierce) at 4M-BM-0C overnight to prepare antibody-immobilized beads. 20 million cells were harvested and washed four times with ice-cold DEPC-treated PBS. The cell pellet was lysed with 500 M-NM-<l of lysis buffer and the supernatant was incubated with Protein G agarose beads without antibody to reduce nonspecific adsorption. The cell lysate was then transferred into a tube containing antibody-immobilized Protein G agarose beads and incubated for 3 hours at 4M-BM-0C. Genome-wide expression levels were analyzed in total RNA samples from total cell lysate and antibody-immobilized Protein G agarose beads-RNP complexes from cells transfected with miR-202 mimic or negative control using the Agilent 44K 60-mer human whole-genome microarray. Signal hybridization and scans were performed by MOGene, LC (St Louis, MO) (run in biological duplicate). The normalized signal intensities of probes from RNP-bead complexes (post-IP fraction) were divided by the signal intensities from total cell lysate (pre-IP fraction) in miR-202 mimic-transfected cells. Transcripts were identified as members of the global miRNA targetome if they exhibited an enrichment fold change > 2.0. From this list, post-IP to pre-IP signal intensity ratios in miR-202 mimic-transfected cells were divided by post-IP to pre-IP signal intensity ratios in NC cells. Transcripts exhibiting normalized signal ratios of > 1.5 were considered to be bound with miR-202 in the RNA-induced silencing complex (RISC), and therefore potential direct miR-202 targets.
2012-12-19 | E-GEOD-42981 | biostudies-arrayexpress