Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human umbilical vein cells under/over expressing mir-210

ABSTRACT: MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. Previously, we demonstrated that miR-210 is a key player of endothelial cell (EC) response to hypoxia, modulating EC survival, migration and ability to form capillary like-structures. Moreover, the receptor tyrosine kinase ligand Ephrin-A3 was identified as one functionally relevant target. Since each miRNA regulates hundreds of mRNAs, different approaches were combined to identify new miR-210 targets: a Using target prediction software, 32 new miR-210 potential targets were identified. b The proteomic profiling of miR-210 over-expressing ECs identified 11 proteins that were specifically inhibited by miR-210, either directly or indirectly. c Affymetrix based gene expression profiles identified 51 genes that were both down-modulated by miR-210 over-expression and de-repressed when miR-210 was blocked. Surprisingly, only few genes identified either by proteomics or transcriptomics were recognized as miR-210 targets by target prediction algorithms. However, a low-stringency pairing research revealed enrichment for miR-210 putative binding sites, raising the possibility that these genes were targeted via non-canonical recognition sequences. To clarify this issue, miR-210-loaded RISC was purified by immuno-precipitation along with its mRNA targets. The presence of Ephrin-A3 mRNA in the complex validated this approach. We found that 32 potential targets were indeed enriched in miR-210-loaded RISC, and thus can be considered as genuine miR-210 targets. In keeping with this conclusion, we were able to further validate a sub-set of them by 3âUTR-reporter assays. Gene ontology analysis of the targets confirmed the known miR-210 activity in differentiation and cell cycle regulation, highlighting new functions such as involvement in RNA processing, DNA binding, development, membrane trafficking and amino acid catabolism. In conclusion, we validated a multidisciplinary approach for miRNAs target identification and indicated novel molecular mechanisms underpinning miR-210 role in EC response to hypoxia. Experiment Overall Design: Gene expression modifications induced by both miR-210 over-expression and blockade were evaluated. In order to identify new direct and indirect miR-210 targets, transcripts repressed by miR-210 over-expression and up-regulated by miR-210 inhibition (and vice versa) were selected.

ORGANISM(S): Homo sapiens

SUBMITTER: Mario Pescatori

PROVIDER: E-GEOD-16962 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:MicroRNAs (miRNAs) are small non-protein-coding RNAs that are incorporated into the RNA-induced silencing complex (RISC) and inhibit gene expression by regulating the stability and/or the translational efficiency of target mRNAs. Previously, we demonstrated that miR-210 is a key player of endothelial cell (EC) response to hypoxia, modulating EC survival, migration and ability to form capillary like-structures. Moreover, the receptor tyrosine kinase ligand Ephrin-A3 was identified as one functionally relevant target. Since each miRNA regulates hundreds of mRNAs, different approaches were combined to identify new miR-210 targets: a Using target prediction software, 32 new miR-210 potential targets were identified. b The proteomic profiling of miR-210 over-expressing ECs identified 11 proteins that were specifically inhibited by miR-210, either directly or indirectly. c Affymetrix based gene expression profiles identified 51 genes that were both down-modulated by miR-210 over-expression and de-repressed when miR-210 was blocked. Surprisingly, only few genes identified either by proteomics or transcriptomics were recognized as miR-210 targets by target prediction algorithms. However, a low-stringency pairing research revealed enrichment for miR-210 putative binding sites, raising the possibility that these genes were targeted via non-canonical recognition sequences. To clarify this issue, miR-210-loaded RISC was purified by immuno-precipitation along with its mRNA targets. The presence of Ephrin-A3 mRNA in the complex validated this approach. We found that 32 potential targets were indeed enriched in miR-210-loaded RISC, and thus can be considered as genuine miR-210 targets. In keeping with this conclusion, we were able to further validate a sub-set of them by 3’UTR-reporter assays. Gene ontology analysis of the targets confirmed the known miR-210 activity in differentiation and cell cycle regulation, highlighting new functions such as involvement in RNA processing, DNA binding, development, membrane trafficking and amino acid catabolism. In conclusion, we validated a multidisciplinary approach for miRNAs target identification and indicated novel molecular mechanisms underpinning miR-210 role in EC response to hypoxia.

Project description:This study was designed to explore the role of miRNA-146a (miR-146a) and its target genes in the endothelial cells. In this study we have demonstrated that lipopolysaccharide (LPS) induced upregulation of miR-146a in the human umbilical vein endothelial cells (HUVEC) and the induction was blocked by the silencing of the toll-like receptors (TLRs) adaptor molecules MyD88 and nonspecific NF-M-NM-:B inhibitor BAY 11-7082. In addition, knockdown of miR-146a by transfection of the locked nucleic acid (LNA)-antimiR-146a significantly decreased the increased cell migration and tube formation induced by LPS. A combined analysis of the bioinformatics miRanda algorithms and the whole genome expression microarray of the immunoprecipitated Ago2 ribonucleoprotein complex identified 14 transcripts as the potential target genes. Subsequent transfection with miR-146a precursor pre-miR-146a into the HUVEC validated that the CARD10 was the target gene of the miR-146a both in the transcript and protein level. immunoprecipitation of RNA-induced silencing complexes (RISC) with an Ago2-specific monoclonal antibody followed by RNA extraction and subsequent quantification of mRNAs on microarrays was utilized to identify mRNAs that are associated with the RNA-silencing machinery and are therefore targets of cellular miRNAs. In brief, 100 M-NM-<g of total protein was diluted with 200 M-NM-<L of PBS buffer (pH 7.4). For each sample, 25 M-NM-<L of Protein A/G plus agarose (Santa Cruz, Dallas, TX) was washed with PBS and incubated with 2 M-NM-<g of rabbit anti-Ago2 (Abcam, Cambridge, MA) or rabbit normal IgG (Santa Cruz) antibodies for 2 h at 4M-BM-0C. The beads containing the immobilized anti-Ago2 antibody were then added to 400 M-NM-<L of diluted serum and incubated for 4 h at 4M-BM-0C. The beads were washed 3 times with 1% NT-2 buffer (1% Nonidet P-40, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 2 mM EDTA) and the mixture was split in half. Each sample was eluted in 600 M-NM-<L of lysis/binding buffer for total RNA extraction and subsequent whole genome array experiment. Microarray analyses. Microarray experiments were performed on the total RNA samples from the Ago2 immunoprecipitate (pooling from n=4 in each group) from 1M-CM-^W106 HUVEC with or without 1 M-NM-<g/mL LPS treatment for 24 h in the presence or absence of miR-146a knockdown by LNA-antimiR-146a or LNA-control, respectively. The procedures were carried out following the manufacturerM-bM-^@M-^Ys protocols. Briefly, 0.5 M-NM-<g of total RNA was amplified by a Fluorescent Linear Amplification Kit (Agilent Technologies) and labeled with Cy3-CTP or Cy5-CTP (PerkinElmer, Waltham, MA) during the in vitro transcription process. RNA from the experimental groups and control group was labeled by Cy5 and Cy3, respectively. A total of 0.825 M-NM-<g of Cy-labled cRNA was fragmented to an average size of about 50-100 nucleotides by incubation with fragmentation buffer (Agilent Technologies) at 60oC for 30 minutes. Correspondingly fragmented labeled cRNA is then pooled and hybridized to Agilent Whole Human Genome 4x44k oligo microarray (Agilent Technologies) at 60M-BM-0C for 17 h. After washing and drying by nitrogen gun blowing, microarrays are scanned with an Agilent microarray scanner (Agilent Technologies) at 535 nm for Cy3 and 625 nm for Cy5. Scanned images are analyzed by Feature extraction software 9.5.3 (Agilent Technologies), an image analysis and normalization software is used to quantify signal and background intensity for each feature, substantially normalized the data by rank-consistency-filtering LOWESS method.

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Transcription profiling of human umbilical vein cells under/over expressing mir-210

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