MiR-34a, miR-122, miR-206 and miR-210 targets identification
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ABSTRACT: To identify putative novel specific targets of miR-34a, miR-122, miR-206 and miR-210, we overexpressed these miRNAs in human Hela cells by transfecting them with synthetic pre-miRNAs or a synthetic “negative” pre-miRNA as control (miR-Neg1). RNA samples were harvested at 48 hours post-transfection and 2 independent experiments were carried out.
Project description:Specificity of interaction between a microRNA (miRNA) and its targets crucially depends on the seed region located in its 5’-end. It is often implicitly considered that two miRNAs sharing the same biological activity should display similarity beyond the strict six nucleotide region that forms the seed, in order to form specific complexes with the same mRNA targets. We have found that expression of hsa-miR-147b and hsa-miR-210, though triggered by different stimuli (i.e. lipopolysaccharides and hypoxia, respectively), induce very similar cellular effects in term of proliferation, migration and apoptosis. Hsa-miR-147b only shares a “minimal” 6-nucleotides seed sequence with hsa-miR-210, but is identical with hsa-miR-147a over 20 nucleotides, except for one base located in the seed region. Phenotypic changes induced after heterologous expression of miR-147a strikingly differ from those induced by miR-147b or miR-210. In particular, miR-147a behaves as a potent inhibitor of cell proliferation and migration. These data fit well with the gene expression profiles observed for miR-147b and miR-210, which are very similar, and the gene expression profile of miR-147a, which is distinct from the two others. Bioinformatics analysis of all human miRNA sequences indicates multiple cases of miRNAs from distinct families exhibiting the same kind of similarity that would need to be further characterized in terms of putative functional redundancy. Besides, it implies that functional impact of some miRNAs can be masked by robust expression of miRNAs belonging to distinct families. To compare the set of transcripts targeted by hsa-miR-147a, hsa-miR-147b and hsa-miR-210, we overexpressed these miRNAs in human lung adenocarcinoma A549 cells by transfecting them with synthetic pre-miRNAs or a synthetic “negative” pre-miRNA as a control (miR-Neg). RNA samples were harvested at 48 hours post-transfection and 3 independent experiments were carried out. 48 hours post-transfection, 3 independent experiments were performed in dye-swap: hsa-miR-147a versus miR-Neg; hsa-miR-147b versus miR-Neg; hsa-miR-210 versus miR-Neg.
Project description:MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of nearly all biological processes. Global miRNA biogenesis is altered in many cancers and RNA-binding proteins (RBPs) have been shown to play a role in this process, presenting a promising avenue for targeting miRNA dysregulation in disease. miR-34a exhibits tumor-suppressive functions by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor Bcl-2, among other regulatory pathways such as Wnt, TGF-, and Notch signaling. Many cancers show downregulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo; however, the post-transcriptional mechanisms by which miR-34a is lost in cancer are not entirely understood. Here, we have used a proteomics-mediated approach to identify Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RBP with no previously reported role in miRNA regulation. We demonstrate that SART3 binds pre-miR-34a with specificity over pre-let-7d and begin to elucidate a new functional role for this protein in non-small lung cancer cells. Overexpression of SART3 led to increased miR-34a levels, downregulation of the miR-34a target genes CDK4 and CDK6, and cell cycle arrest in the G1 phase. In vitro binding studies showed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Collectively, our results present evidence for an influential role of SART3 in miR-34a biogenesis and cell cycle progression.
Project description:To identify putative fibroblasts-specific targets of mir-155, we overexpressed mir-155 in lung fibroblasts by transfecting them with a synthetic pre-mir-155 or a synthetic “negative” pre-miRNA as control (miR-Neg). RNA samples were harvested at 24 and 48 hours post-transfection and 2 independent experiments were carried out.
Project description:To identify putative novel specific targets of mir-199-5p, we overexpressed miR-199a-5p as well as miR-21 and a siRNA targeted against CAV1 in human HFL1 pulmonary fibroblasts (CCL-153) by transfecting them with synthetic pre-miRNAs or a synthetic “negative” pre-miRNA as control (miR-Neg). RNA samples were harvested at 48 hours post-transfection and 2 independent experiments were carried out. Additional samples correspond to HFL1 cells treated or not with 10ng/ml TGFbeta for 48 hours in the absence of serum (2 independent experiments).
Project description:To identify putative novel specific targets of mir-199-5p, we overexpressed miR-199a-5p as well as miR-21 and a siRNA targeted against CAV1 in human HFL1 pulmonary fibroblasts (CCL-153) by transfecting them with synthetic pre-miRNAs or a synthetic “negative” pre-miRNA as control (miR-Neg). RNA samples were harvested at 48 hours post-transfection and 2 independent experiments were carried out. Additional samples correspond to HFL1 cells treated or not with 10ng/ml TGFbeta for 48 hours in the absence of serum (2 independent experiments). 2 independent experiments performed in a one color design, corresponding to 7 conditions (miR-Neg, miR-199-5p, miR-21, si-Neg, si-CAV1, control, TGFbeta) and a total of 14 samples.
Project description:To identify putative novel specific targets of mir-210, we overexpressed miR-210 as well as miR-34a and a siRNA targeted against E2F3 in A549 human adenocarcinoma cells by transfecting them with synthetic pre-miRNAs or a synthetic “negative” pre-miRNA as control (miR-Neg). RNA samples were harvested at 48 hours post-transfection and 2 independent experiments were carried out.
Project description:To identify putative novel specific targets of miR-199-5p, miR-199a-3p and miR-214-3p, we overexpressed these miRNAs in human MRC5 pulmonary fibroblasts (CCL-171) using synthetic pre-miRNAs or a synthetic “negative” pre-miRNA control (miR-Neg). RNA samples were harvested 48 hours post-transfection and 3 independent experiments were carried out.
Project description:To identify putative novel specific targets of mir-210, we overexpressed miR-210 as well as miR-34a and a siRNA targeted against E2F3 in A549 human adenocarcinoma cells by transfecting them with synthetic pre-miRNAs or a synthetic “negative” pre-miRNA as control (miR-Neg). RNA samples were harvested at 48 hours post-transfection and 2 independent experiments were carried out. 48 hours post-transfection, 2 independent experiments performed in dye-swap: miR-210 versus miR-Neg ; miR-34a versus miR-Neg ; si-E2F3 versus miR-Neg ; si-control versus miR-Neg.
Project description:To identify putative novel specific targets of miR-141-3p, we overexpressed this miRNAs in primary keratinocytes using a synthetic mimic (pre-miR-141-3p) or a synthetic “negative” control mimic (pre-miR-ctrl). RNA samples were harvested 30 hours post-transfection and 3 independent experiments were carried out.
Project description:To identify putative novel specific targets of miR-203-3p, we overexpressed this miRNAs in primary keratinocytes using a synthetic mimic (pre-miR-203a-3p) or a synthetic “negative” control mimic (pre-miR-ctrl). RNA samples were harvested 30 hours post-transfection and 3 independent experiments were carried out.