Differentiation of IMR32 neuroblastoma is accompanied by a global change in the transcriptome
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ABSTRACT: The aim of this study is to compare human transcriptomes based on NGS (RNA-seq). The transcriptome of neuroblastoma IMR32 was compared with the transcriptome of neuroblastoma IMR32, which was differentiated for 16 days in the presence of 2.5 mkM BrdU. Methods. Human mRNA profiles of 16-day differentiation of IMR32 neuroblastoma and non-differentiated IMR32 neuroblastoma were obtained by deep three-fold sequencing using Illumina NovaSeq. The mapping is read into the human genome (hg38) using the hisat program. On average, about 89-90% of all data received was unambiguously aligned in each library. The htseq-count utility has counted the number of reads that have been matched against known genes (ncbi - entrezID). The obtained values (cpm - countpermillion) for each gene for each library were combined into one matrix for further analysis. Results: Using an optimized data analysis workflow, we matched about 30 million sequence reads per sample to the human genome (hg38). Filtration, normalization by the method (TMM), variance estimation and differentially expressed genes estimation were performed in the edgeR module. Genes in which the cpm did not exceed 1 in any three libraries were considered low expressing.
Project description:The aim of this study is to compare the NGS-derived profiling of human transcriptome (RNA-seq) in wild-type neuroblastoma cells with neuroblastoma transcript with a high level of expression of Oct-1 transcription factor and evaluate the effect of Oct-1 transcription factor on the regulation of nerve cell differentiation. Methods: Human mRNA profiles of 16-day differentiating wild-type neuroblastoma IMR32 and neuroblastoma IMR32 with overexpression primate-specific isoform of transcription factor Oct-1 were generated by deep sequencing, in triplicate, using Illumina NovaSeq. Mapping reads to the human genome (hg38) using hisat program. On average, about 89-90% of all received data was uniquely aligned in each library. The htseq-count utility calculated the number of reads that were mapped to known genes (ncbi - entrezID). The obtained values (cpm - countpermillion) for each gene for each library were combined into one matrix for further analysis. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the human genome (hg38). Filtration, normalization by the method (TMM), variance estimation and evaluation of differentially expressed genes were performed in the edgeR module. Genes in which cpm did not exceed 1 in any three libraries were considered low-expressing. After filtering the low-expressing genes, 15,108 entries remained. RNA-Seq data confirmed that approximately 8% of the transcripts showed differential expression between the WT and Oct-1 overexpressed differentiating neuroblastoma cells, with FDR<0.01 (p-value adjusted for multiple testing).
Project description:Transcriptional profiling of neuroblastoma cell line expressing the PML1 isoform. Two-condition experiment: PML1 IMR32 vs empty vector IMR32. Two biological replicates, dye-swapped.
Project description:The goal of this study was to look at genes that were affected by 69-kDa and/or 82-kDa ChAT proteins in IMR32 cells Experiment Overall Design: The gene expression changes of IMR32 cells stably expressing either 69-kDa or 82-kDa ChAT proteins were anaylzed and compared to control IMR32 wild type cells. 3 biological replicates were anaylzed per condition (69-kDa ChAT expressing cells, 82-kDa ChAT expressing cells, or wild type IMR32 cells) for a total of 9 samples altogether.
Project description:Neuroblastoma is an embryonal tumor which originates from neural crest progenitor cells that fail to differentiate along their predefined route to sympathetic neurons or sympatho-adrenergic adrenal cells. It is the most common extracranial tumor of childhood and accounts for 15% of all childhood cancer deaths. Especially patients suffering from high grade or relapsed neuroblastoma have poor outcome in spite of aggressive treatment regimens including autologous stem cell transplantation. Those patients are in urgent need of additional effective therapies which demands the development of targeted approaches. Dihydroorotatedehydrogenase (DHODH) is the fourth enzyme of the pyrimidine synthesis pathway which oxidizes dihydroorotate to orotate. In recent past it became a potential drug target for cancer treatment because of its keyrole in processing essential pyrimidine nucleotides. On the basis of the existing data, functional inhibition of DHODH is considered to be promising therapeutic option for several tumor entities like advanced colorectal, breast or lung-cancers. Leflunomide is an established drug in treatment of the autoimmune diseases rheumatoid arthritis and multiple sclerosis. In the liver Leflunomide becomes converted to its active metabolite called Teriflunomide, which inhibits the activity of DHODH directly. In recent times Leflunomide is also used for therapy against the Cytomegalovirus and the BK virus. Also for Melanoma was shown recently a decreased growth rate due to Leflunomide treatment in a zebrafish and a mouse model. As Melanoma is a malignant tumor of the skin, which derives also from neural crest progenitor cells, a coherent investigation of effictivity of Leflunomide in neuroblastoma celllines showed first promising results. The aim of our study was to reanalyse the effectivity of Leflunomide in Neuroblastoma and to shed further light in its biological mode of action. Three+B52 biological samples of the human neuroblastoma cell line IMR32 were treated with DMSO or Teriflunomide [118 ᅡᄉM]
Project description:IMR32 was transduced with control or shMYCN virus (duplo). For each experiment 3 time points were analyzed and 2 no virus controls. IMR32 was transduced with control or shMYCN virus (duplo). For each experiment 3 time points were analyzed and 2 no virus controls.