Project description:Here we studied the role of oxidized phospholipids in mediating phenotype switching of endothelial cells between quiescent and angiogenic states. Two oxPAPC datasets, a microRNA array and global run-on sequencing (GRO-seq), was combined with Nuclear factor erythroid 2-Related Factor 2 (NRF2) binding model to select candidate miRNAs for further studies. The pre-screening resulted in a selection of miR-106b~25 cluster for further studies. The cluster was shown to be both oxPAPC-responsive and NRF2-regulated, and its diagnostic and prognostic potential was investigated in pericardial fluid samples of heart failure and atherosclerosis patients. As the most abundant member of the cluster in both endothelial cells and pericardial fluid of atherosclerosis patients, miR-93-5p was selected for more detailed studies. RNA-seq from miR-93 overexpressing cells revealed significant changes in pathways related to angiogenesis. Together with NRF2, miR-93 was shown to control endothelial plasticity through regulation of the key players, namely Krüppel-like factor 2 (KLF2) for quiescence, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) for glycolysis, and Vascular Endothelial Growth Factor A (VEGFA), Forkhead box protein O1 (FOXO1) and MYC proto-oncogene protein (MYC) for growth and proliferation.The findings show that NRF2 and miR-93 control the activity of endothelial cells and mediate the effects of oxPAPC on endothelial activation, collectively providing novel mechanisms for the control of endothelial plasticity and oxPAPC response.
Project description:Sepsis remains a leading cause of death and currently has no pathogenesis-specific therapy. Hampered progress is partly due to the lack of insight into deep mechanistic processes. Deciphering the functions of microRNAs in sepsis pathogeny became a dynamic research topic in the last decade. To screen for new microRNA targets for sepsis therapeutics, we used human samples: microRNA array data from peripheral blood mononuclear cells (PBMCs) from sepsis patients and controls, whole blood samples from sepsis survivors; and multiple animal models: mouse cecum ligation-puncture (CLP)-induced sepsis, mouse viral microRNA challenge, and baboon Gram-positive and Gram-negative sepsis models. miR-93-5p met the criteria for a therapeutic target, being overexpressed in baboons which died early after induction of sepsis and downregulated in humans who survived after sepsis. Therapeutically, inhibiting miR-93-5p prolonged the overall survival of mice with CLP-induced sepsis, with a stronger effect in old mice. Mechanistically, anti-miR-93-5p therapy reduced inflammatory monocytes and increased circulating effector memory T cells, especially CD4+ subset. Ago2-immunoprecipitation in miR-93-knockout T cells identified important regulatory receptors, CD28 and CD160, as direct miR-93-5p target genes. In conclusion, miR-93-5p is a potential therapeutic target in sepsis in the elderly through its regulation of both innate and adaptive immunity. We applied the Ago2-RIP Chip (Ribonucleoprotein ImmunoPrecipitation followed by microarray analysis) to identify miR-93-5p target mRNAs. This methodology uses beads coupled to either anti-Ago2 antibody (or an IgG control) to pull down the RISC complex together with any miRNA-interacting mRNAs. To define target genes of miR-93-5p, we assessed mRNA transcripts enriched in the Ago2-IP fraction of JURKAT parental cells or control cells (where miR-93-5p is present) but not in the two KO clones (where miR-93-5p is absent) by microarray
Project description:Purpose: The goals of this study are to compare the transcriptome profiling among LEPC-Control, LEPC-Biotin-miR-93-5p and LEPC-Biotin-miR-93-5p-Muttion group to elevate the enrichment mRNA by Biotin-miR-93-5p in LEPC cells. Methods: LEPC-Control, LEPC-miR-93-5p and LEPC-miR-93-5p-MDX cells mRNA profiles were generated by deep sequencing, in triplicate, using Illumina Hiseq2500/Miseq. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. Results: we set the increase by ≥ 4 fold and transcripts at an FPKM ≥1 in LEPC cells as significance. By stepwise analysis, we identifies 615 high confident miR-93-5p-targted mature mRNA that is selectively enriched by biotin-miR-93-5p, but not biotin-miR-93-5p mutant, the miR-93-5p targetome we identified by HITS-CLSBP has 97.4% overlap with online predicted miR-93-5p mRNA targets. Conclusions: Overexpression of miR-93-5p instigates the malignant transformation of LEPC by regulating multiple cancer-related pathways.
Project description:MiRNAs have been shown to alter both protein expression and secretion in different cellular contexts. By combining in vitro, in vivo and in silico techniques, we demonstrated that overexpression of pre-miR-1307 reduced the ability of breast cancer cells to induce endothelial cell sprouting and angiogenesis. However, the molecular mechanism behind this and the effect of the individual mature miRNAs derived from pre-miR-1307 on protein secretion and is largely unknown. Here, we overexpressed miR-1307-3p|0, -3p|1 and 5p|0 in MDA-MB-231 breast cancer cells and assessed the impact of miRNA overexpression on protein secretion by Mass Spectrometry. Unsupervised hierarchical clustering revealed a distinct phenotype induced by overexpression of miR-1307-5p|0 compared to the controls and to the 5’isomiRs derived from the 3p-arm. Together, our results suggest different impacts of miR-1307-3p and miR-1307-5p on protein secretion which is in line with our in vitro observation that miR-1307-5p, but not the isomiRs derived from the 3p-arm reduce endothelial cell sprouting in vitro. Hence these data support the hypothesis that miR-1307-5p is at least partly responsible for impaired vasculature in tumors overexpressing pre-miR-1307.
Project description:Myocardial regeneration is restricted to early postnatal life, when mammalian cardiomyocytes still retain the ability to proliferate. The molecular cues that induce cell cycle arrest of neonatal cardiomyocytes towards terminally differentiated adult heart muscle cells remain obscure. We report that the miR-106b~25cluster is higher expressed in the early postnatal myocardium and decreases in expression towards adulthood, especially under conditions of overload, and orchestrates the transition of cardiomyocyte hyperplasia towards cell cycle arrest and hypertrophy by virtue of its targetome. To identify the relevant targets of individual miRNAs in the miR-106b~15 cluster and elucidate the molecular mechanisms underlying the proliferative effects of this microRNA cluster, we assessed the global transcriptomic changes by deep-sequencing total neonatal mouse cardiomyocyte RNA after exogeneous transfection with hsa-miR-106b-5p, hsa-miR-93-5p, hsa-miR-25-3p and compared the transcriptomic profiles to cardiomyocytes transfected with cel-miR-67, a control miRNA.
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.
Project description:This is a prospective-retrospective study to determine if the expression of the miRNA’s miR-31-3p and miR-31-5p are prognostic of patient outcomes or predictive of the benefit from anti-EGFR therapy in stage III Colon Cancer. The present study will utilize FFPE tumor samples collected from patients enrolled in the PETACC-8 study conducted by the Fédération Francophone de Cancérologie Digestive (FFCD). This phase 3 clinical trial prospectively randomized fully resected stage III colon cancer patients to receive adjuvant treatment with either FOLFOX-4 plus cetuximab or FLOFOX-4 alone.
Project description:The transition of the endothelium to a pro-inflammatory state is key to progression of chronic inflammatory diseases including rheumatoid arthritis, chronic bowel disease and atherosclerosis. In atherosclerosis it is hypothesized that low density lipoproteins (LDL) that become trapped in the intima of the blood vessels are oxidized to minimally modified LDL (mmLDL) and that these serve as an important contributing factors to endothelial dysfunction. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine (OX-PAPC), a model of the active phospholipid components of mmLDL affects the expression of hundreds of genes involved in inflammatory and other biological processes in human aortic endothelial cells (HAECs). We hypothesized that microRNAs (miRNAs) partially regulate this response. Using next generation sequencing, we identified miR-21-3p and miR-27a-5p to be induced 4-fold and 3-fold, respectively in response to OX-PAPC treatment compared to control treatment in HAECs. To identify the targets, we performed whole genome transcript profiling following transient over-expression of these two miRNAs followed by. In total, 1254 genes were down-regulated with 925 of them overlapping between the two miRNAs. Functional enrichment analysis using Gene Ontology predicted that the two miRNAs were involved in the regulation of NF-κB signaling. We characterized the Toll/interleukin-1 receptor (TIR) domain-containing adaptor protein TICAM2 as a direct target of miR-21-3p and miR-27a-5p. Furthermore, we showed that over-expression of miR-21-3p and miR-27a-5p lead to decreased p65 translocation to the nucleus and decreased the expression of known NF-κB downstream target genes confirming both miRNAs’ role in negatively regulating NF-κB signaling in endothelial cells. mRNA expression profiling of human aortic endothelial cells from two separate donors that were transfected with 1 nM microRNA mimics and negative control. The miRIDIAN mimics used were miR-21-3p (Catalog Number:C-301023-01-0005), miR-27a-5p (Catalog No: C-301028-01-0005), negative control (Catalog No: CN-001000-01-05)
Project description:miR-93 and 135b were transfected at 50nM in mouse embryonic fibroblasts (MEFs). Total RNAs were extracted at 48hrs post transfection and mRNA expression was then analyzed.