Project description:We aimed to characterize decoy to the RNA-binding protein CUG-RNA binding protein 1 (CUGBP1 mechanism in A549 lung cancer cells. We identified several new canonical targets of CUGBP1 but those were not regulated by miR-574-5p via the decoy mechanism. This can be explained by the localization of CUGBP1 and miR-574-5p in the nucleus, where CUGBP1 regulates alternative splicing. Next, we analyzed the 3’UTRs of potential targets and found that the decoy-dependent regulation of mPGES-1 splicing is unique. Therefore, we postulate that in A549 cells mPGES-1 is the only protein regulated by the decoy mechanism of CUGBP1 and miR-574-5p which suggests that the decoy mechanism allows the specific regulation of the expression of distinct targets.
Project description:The identification of miRNAs’ targets and associated regulatory networks might allow the definition of new strategies using drugs whose association might mimic a given miRNA’s effects. Based on this assumption our group devised a multi-omics approach in an attempt to precisely characterize miRNAs’ effects. We combined the analysis of miR-491-5p direct targets, and effects at the transcriptomic and proteomic levels. We thus constructed an interaction network which enlightened highly connected nodes, being either direct or indirect targets of miR-491-5p effects: the already known EGFR and BCL2L1, but also EP300, CTNNB1 and several small-GTPases. By using different combinations of specific inhibitors of these nodes, we could greatly enhance their respective cytotoxicity and mimic miR-491-5p-induced phenotype. Our methodology thus constitutes an interesting strategy to comprehensively study the effects of a given miRNA. Also, we identified targets for which pharmacological inhibitors are already available for a clinical use, or in clinical trial phases. This study might thus enable innovative therapeutic options for ovarian cancer, which remains the first cause of death from gynecological malignancies in developed countries.
Project description:While microRNAs (miRs) have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T cell activation are less clear. We found reduced levels of miR-15a/16 at 3-18 h post-T cell receptor (TCR) stimulation, suggesting a role in shaping T cell activation. An inducible miR15a/16 transgenic mouse model was developed to determine how elevating miR-15a/16 levels during early stages of activation would affect T cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycyclin (DOX) induced expression of miR-15a/16 from 0-18 h post-TCR stimulation decreased ex vivo proliferation as well as in vivo antigen-specific proliferation. Bioinformatic and proteomic approaches were combined to identify MEK1 as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased MEK1 containing the 3’-UTR target nucleotide sequence (UGCUGCUA) but did not decrease MEK1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules ERK1/2 and Elk1 were decreased with DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T cell activation to facilitate increased MEK1 and ERK1, and this promotes sustained MEK1-ERK1/2-Elk1 signaling required for optimal proliferation.
Project description:Hemopoiesis entails a series of hierarchically organized events that proceed throughout cell specification and terminates with cell differentiation. Commitment needs the transcription factors effort that, in concert with microRNAs, drives cell fate specification, answering to promiscuous patterns of gene expression by turning on lineage-specific genes and repressing alternate lineage transcripts. Therefore microRNAs and mRNAs cooperate to direct cell fate decisions. We obtained microRNAs profiles from human CD34+ hemopoietic progenitor cells and in-vitro differentiated erythroblasts, megakaryoblasts, monoblasts and myeloblasts precursors and we analyzed them together with the gene expression profiles of the same populations. We found that for most part of microRNAs specifically up-regulated in one single cell progeny an inverse correlation between microRNAs and down-regulated putative targets expression levels occurs. We chose hsa-mir-299-5p as a model to get further insights into the possible biological relevance of this microRNAs-mRNAs expression integrated analytical approach and we asked if the forced expression of a single lineage-specific microRNA is able to control the cell fate of CD34+ progenitors grown in multilineage culture conditions. Gain and loss of-function experiments established that mir-299-5p regulates hemopoietic progenitors fate modulating reciprocally megakaryocytic-granulocytic versus erythroid-monocytic differentiation and has at least two genuine targets, the transcription factors CTCF and SOX4. CD34+ hematopoietic progenitor cells were transfected with the Amaxa Nucleofector Device, using the Human CD34 Cell Nucleofection Kit, accordingly to the manufacturer’s instructions (Amaxa Biosystem, Cologne, Germany), and 5µg of either the Pre-miR miRNA Precursor Molecule—Negative Control # 1 (NC1) or the hsa-mir-299-5p Pre-miR miRNA Precursor Molecule (299-5p) (Ambion, Austin, TX, USA) and pulsed with the program U-008. The dataset is composed of three independent paired experiment of 299-5p gain of-function (three hsa-299-5p Pre-miR miRNA Precursor Molecule nucleoporated samples and three paired Pre-miR miRNA Precursor Molecule—Negative Control # 1 transfected ones).
Project description:mRNA microarray experiments were performed to measure global mRNA expression in the presence of increased or decreased miR-106a-5p levels to to identify the total transcripts regulated by miR-106a-5p directly or indirectly. FASTK was identified as a direct target gene of miR-106a-5p. In order to identify the total transcripts regulated by miR-106a-5p directly or indirectly, we first measured the global mRNA expression change through mRNA microarray by overexpressing or knocking down miR-106a-5p in cancer cells. Next, we combined bioinformatics programs to select candidate miR-106a-5p targets from the differentially regulated genes to refine the number of miR-106a-5p targets. Then we validated the miR-106a-5p target gene through western blot analysis, quantitative real-time PCR and luciferase reporter assay.
Project description:Overexpression of miR-183-5p|+2, but not of the other two isomiRs |0 and |+1, was observed to reduce cell cycle and cell proliferation in different triple-negative breast cancer cell lines. Therefore, we hypothesized that the |+2 isoform has targets distinct from the other two isoforms. To test this hypothesis, we overexpressed separately the three different isoforms or negative controls (siAllstar or mimic-Cltr) and performed Mass Spectrometry to identify differentially regulated proteins. Interestingly, a gene set enrichment analysis of the changes in protein expression revealed significant downregulation of transcriptional targets of E2F specifically in cells transfected with the |+2 isoform prompting us to validate the predicted isomiR specific target E2F1. Subsequently, we could show that direct targeting of E2F1 by miR-183-5p|+2 is responsible for the impact of the isomiR on cell cycle and proliferation.
Project description:Breast cancer (BC) is a commonly identified life-threatening type of cancer and a major cause of death among women worldwide. Several microRNAs (miRs), including miR-143-5p, have been reported to be vital for regulating hallmarks of cancer; however, the effect of miR-143-5p on BC requires further exploration. The present study performed bioinformatics analysis on GSE42072 and GSE41922 datasets from the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database to identify miR-143-5p expression patterns. Furthermore, miR-143-5p expression was detected in BC cell lines and tissues via reverse transcription-quantitative PCR. Post-transfection with miR-143-5p mimics, Cell Counting Kit-8, colony formation and Transwell assays were performed to explore the effects of miR-143-5p on BC cell proliferation, colony formation, and migration. The association of miR-143-5p with the hypoxia-inducible factor-1α (HIF-1α)-associated glucose transporter 1 (GLUT1) pathway was explored via western blotting, immunofluorescence and dual-luciferase reporter assay. The present study detected high expression of miR-143-5p in BC tissue of the GSE42072 and serum of the GSE41922 datasets by GEO chip analysis. Additionally, the expression levels of miR-143-5p were decreased in BC tissues compared with those in adjacent healthy tissues, and low miR-143-5p expression was associated with a poorer prognosis and shorter survival time in patients with BC. In vitro, miR-143-5p expression levels were decreased in BC cells, and transfection with miR-143-5p mimics suppressed BC cell proliferation, colony formation, migration. Furthermore, miR-143-5p targeted the HIF-1α-related GLUT1 pathway, and inhibited HIF-1α and GLUT1 expression. Additionally, HIF-1α agonists reversed the miR-143-5p-induced inhibition during tumorigenesis. In conclusion, miR-143-5p exhibited low expression in BC tissues, and suppressed BC cell proliferation, colony formation, migration. Moreover, the antitumor effects of miR-143-5p targeted the HIF-1α-related GLUT1 pathway.
Project description:To identify target genes of miR-142-5p and miR-130a-3p that are involved in M2 polarization, we examined the mRNA expression profile changes after altering miR-142-5p or miR-130a-3p expression in IL-4-treated macrophages. Macrophages were transfected with control ASO, miR-142-5p ASO, control mimics or miR-130-3p mimics using lentiviral vectors. After 24 hr, the cells were treated with IL-4 for 24h.mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The labeled cRNAs were hybridized onto the Human RNA Array v2.0 (8 x 60K, Arraystar). After having washed the slides, the arrays were scanned by the Agilent Scanner G2505B.