Project description:Platelets from sickle cell disease patients have differentially expressed microRNAs as compared to platelets from healthy control subjects. The objective of this study is to overexpress an upregulated miRNA,miR-1225-3p, in MEG01 cells to identify putative targets. MEG-01 cells were transfected with microRNA-1225-3p mimic for overexpression. Simultaneously, a negative control (scrambled) RNA was transfected for comparison. Gene expression was measured at 24 hours after transfection. Five independent experiments were performed at the same time in each group: microRNA-1225-3p transfected and scrambled.
Project description:GAS2DN could suppress the growth of chronic myeloid leukemia cells, including K562, MEG-01 and CD34+ cells from patients. In addition, GAS2DN inhibited the tumorigenic ability of MEG-01 cells in nude mice. To understand the molecular insight of this inhibitory effect of GAS2DN, global gene expression were performed. The control and GAS2DN-transduced MEG-01 cells were used for microarray analysis.
Project description:GAS2DN could suppress the growth of chronic myeloid leukemia cells, including K562, MEG-01 and CD34+ cells from patients. In addition, GAS2DN inhibited the tumorigenic ability of MEG-01 cells in nude mice. To understand the molecular insight of this inhibitory effect of GAS2DN, global gene expression were performed. The control and GAS2DN-transduced MEG-01 cells were used for microarray analysis. Three biological independent extracts of control and GAS2DN-transduced cells were pooled together with equal amount, and then the pooled samples were compared with Affymetrix chips.
Project description:Epstein-Barr virus is associated with several human malignancies, including Burkitt Lymnphoma. The virus encodes more than 40 microRNAs, which participate in its possible pathogenetic role. We used microarrays to study the effect of the expression of an Epstein-Barr virus-encoded microRNA (ebv-BART6-3p) on the global gene expression profile of Burkitt Lymphoma cell lines. In order to determine the BART6-3p targets, EBV-negative Akata 2A8 or EBV-positive Akata cells were transiently transfected (in duplicates) with synthetic BART6-3p mimic or inhibitor (100 nM; Custom synthesized by Dharmacon- Thermo Scientific, Germany), respectively. For each treatment, a further transfection with corresponding negative control was performed as well (10 nM; I-300145-01, Dharmacon- Thermo Scientific, Germany). The transfection of 5,000,000 cells per treatment was performed by Amaxa nucleofector apparatus (Amaxa, Cologne-Germany), using the program G23 and the transfection solution V according to the manufacturerâs instructions. Transfection efficiency was assessed by means of a further treatment (2µg of pmaxGFP) and detection of both fluorescence and cell viability by flow cytometry. Twenty four hours post-transfection, cells were harvested and RNA was extracted using Trizol, and transfection efficiency was further confirmed by evaluating the expression level of BART6-3p using q-PCR by means of Taqman probes, employing RNU43 as housekeeping miRNA (Applied Biosystems, Cologne, Germany). RNA was further hybridized to HuGene-2.0-st array (Affymetrix, Santa Clara, CA) according to the manufacturer's instructions.
Project description:We report that cellular O-GlcNAcylation levels decline along the course of megakaryocyte (MK) differentiation from human-derived hematopoietic stem and progenitor cells (HSPCs) and that inhibition of O-GlcNAc transferase (OGT), of which catalyzes O-GlcNAcylation, prolongedly decreases O-GlcNAcylation and induces megakaryopoiesis and thrombopoiesis. To gain the better insight into the potential mechanisms underlying platelet regulation by O-GlcNAcylation ,we compared the genome-wide transcription profiles of megakaryblastic MEG-01 cells with decreased O-GlcNAcylation (OGTi) and its control counterpart using RNA sequencing. Gene ontology and enrichment analysis of differentially expressed genes suggest that platelet formation might be regulated through the perturbation in cell adhesion molecules, i.e. integrin-a4 and b7, downstream of O-GlcNAc/c-Myc axis.
Project description:To clarify the gene expression profile in MLE-12 cells transfected with microRNA mimics upon influenza virus infection, we transfected microRNA mimics (mmu-miR-483-3p or Negative control miRNA) into MLE-12 cells and infected A/Puerto Rico/8/1934 (PR8) strain at an MOI of 2 at 24 hours post transfection. RNA was isolated from cells at 12 hours post infection. We found that miR-483-3p transfection down-regulated the genes involved in the innate immunity regulation upon influenza virus infection.
Project description:Proteomic analysis of cell-free supernatants (Secretome) from human platelets (n = 4, donors) and megakariocytes (MEG-01, n = 3 replicates).
Project description:Megakaryocytes accumulate mRNA during their maturation, which is required for the correct spatio-temporal production of cytoskeletal proteins, membranes and platelet-specific granules, and for the subsequent shedding of thousands of platelets per cell. Gene expression profiling identified the RNA binding protein ATAXIN2 (ATXN2) as a putative novel regulator of megakaryopoiesis. ATXN2 expression is high in CD34+/CD41+ megakaryoblasts and sharply decreases upon maturation to megakaryocytes. ATXN2 associates with DDX6 suggesting that it may mediate repression of mRNA translation during early megakaryopoiesis. Comparative transcriptome and proteome analysis on megakaryoid cells (MEG-01) with differential ATXN2 expression identified ATXN2 dependent gene expression of mRNA and protein involved in processes linked to coagulation. Mice deficient for Atxn2 did not display differences in bleeding times, but expression of key surface receptors on platelets, such as ITGB3 (carries the CD61 antigen) and CD31 (PECAM1), were deregulated and platelet aggregation upon specific triggers was reduced.
Project description:Temozolomide kills cancer cells by forming O6-methylguanine (O6-MeG), which leads to apoptosis due to mismatch-repair overload. However, O6-MeG repair by O6-methylguanine-DNA methyltransferase (MGMT) contributes to drug resistance. Genomic profiles of O6-MeG could elucidate how O6-MeG accumulation is influenced by repair mechanisms, but there are no methods to map genomic locations of O6-MeG. Here, we developed an immunoprecipitation- and polymerase-stalling-based method, termed O6-MeG-seq, to locate O6-MeG across the whole genome at single-nucleotide resolution. We analyzed O6-MeG formation and repair with regards to sequence contexts and functional genomic regions in glioblastoma-derived cell lines and evaluated the impact of MGMT transfection. O6-MeG signatures were highly similar to mutational signatures of patients previously treated with temozolomide. Furthermore, MGMT did not preferentially repair O6-MeG with respect to sequence context, chromatin state or gene expression level, however, may protect oncogenes from mutations. Finally, we found an MGMT-independent strand bias in O6-MeG accumulation in highly expressed genes, suggesting an additional transcription-associated contribution to its repair. These data provide high resolution insight on how O6-MeG formation and repair is impacted by genome structure and regulation. Further, O6-MeG-seq is expected to enable future studies of DNA modification signatures as diagnostic markers for addressing drug resistance and preventing secondary cancers.