Genome-wide analysis of miRNA-associated transcriptome profiles in multiple cell models
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ABSTRACT: Schizophrenia-associated miRNA were bidirectionally modulated in HEK-293, HeLa, and SH-SY5Y cell models. Results provide important insights into the current understanding of miRNA function in various cellular environments. Total RNA was obtained from HEK-293, HeLa, and SH-SY5Y cells at 24hrs post-transfection with either synthetic miRNA (miR overexpression) or anti-miR inhibitor (miR inhibition) oligonucleotides.
Project description:Maternally Expressed Gene 3 (MEG3) encodes a lncRNA which is suggested to function as a tumor suppressor. MEG3 functions as a tumor suppressor in hepatoma cells, whose action is mediated by interaction with p53 protein to activate p53-mediated transcriptional activity and influence the expression of partial p53 target genes. Total RNA was isolated from hepatoma cells (HepG2 and SK-Hep-1) transfected with either control or MEG3 overexpression/knockdown constructs for global expression analysis.
Project description:Insulin receptor antagonist S961 induces pancreatic beta cell replication. Microarray anaysis was performed in liver to find genes that may mediate beta cell replication. Mice were treated with either vehicle (PBS) or S961 (10nMol/week) for a week, the total RNA from liver was isolated and then subjected to illumina microarray analysis.
Project description:The transcription factor Egr3 has been shown to have a cell autonomous role in sympathetic nervous system (SNS) development. We utilized microarray analysis to identify potential downstream target genes deregulated with loss of Egr3. Both conditions were in the null Bax background to prevent apoptosis and therefore mitigate identification of apoptosis related genes. Our analysis identified genes involved in biological processes that were expected such as SNS development and axonogenesis as well as those that were unexpected such as dendritogenesis and axon guidance. This led us to investigate whether Egr3 is important in these unexpected biological processes within sympathetic neurons. Total RNA was obtained from superior cervical ganglion (SCG) dissected from P0 mice with the genotype of Egr3+/+; Bax-/- or Egr3-/-; Bax-/-. Each genotype had 3 samples each.
Project description:Genome-wide expression array measurements for 9 head and neck squamous cell carcinomas (HNSCC) stratified by worst pattern of invasion (WPOI) Jayakar et al. (2016). Apolipoprotein E promotes invasion in oral squamous cell carcinoma. Li et al. (2013). Validation of the risk model: high-risk classification and tumor pattern of invasion predict outcome for patients with low-stage oral cavity squamous cell carcinoma. Comparison of transcription profiles between OSCC tumors with a more invasive (WPOI 5) versus a less invasive (WPOI 3) pattern of invasion using two independent Illumina platforms.
Project description:Glioblastoma (GBM) is thought to be driven by a sub-population of cancer stem cells (CSCs) that self-renew and recapitulate tumor heterogeneity, yet remain poorly understood. Here we present a comparative epigenomic analysis of GBM CSCs that reveals widespread activation of genes normally held in check by Polycomb repressors. These activated targets include a large set of developmental transcription factors (TFs) whose coordinated activation is unique to the CSCs. We demonstrate that a critical factor in the set, ASCL1, activates Wnt signaling by repressing the negative regulator DKK1. We show that ASCL1 is essential for maintenance and in vivo tumorigenicity of GBM CSCs. Genomewide binding profiles for ASCL1 and the Wnt effector LEF1 provide mechanistic insight and suggest widespread interactions between the TF module and the signaling pathway. Our findings demonstrate regulatory connections between ASCL1, Wnt signaling and collaborating TFs that are essential for the maintenance and tumorigenicity of GBM CSCs. Epigenomic profiling of glioblastoma stem cells
Project description:We broadly profiled DNA methylation in breast cancers (n=351) and benign parenchyma (n=47) for correspondence with disease phenotype using formalin-fixed paraffin-embedded (FFPE) diagnostic surgical pathology specimens. Exploratory analysis revealed a distinctive primary invasive carcinoma subclass featuring extreme global methylation deviation. Subsequently we tested the correlation between methylation remodeling pervasiveness and malignant biology. A methyl deviation index (MDI) was calculated for each lesion relative to terminal ductal-lobular unit (TDLU) baseline, and group comparisons revealed that high-grade, short-survival ER+ cancers manifest significantly higher MDI than low-grade, long-survival ER+ cancers. In contrast, ER- cancers display significantly lower MDI, revealing a striking epigenomic distinction between cancer hormone receptor subtypes. Kaplan-Meier survival curves of MDI-based risk classes showed significant divergence between low- and high-risk groups. MDI showed superior prognostic performance to crude methylation levels, and MDI retained prognostic significance (p<0.01) in Cox multivariate analysis including clinical stage and pathologic grade. Most MDI targets individually are significant markers of ER+ cancer survival. Lymphoid and mesenchymal indices were not substantially different between ER+ and ER- groups, and do not explain MDI dichotomy. However, mesenchymal index was associated with ER+ cancer survival, and high lymphoid indices with medullary carcinoma. Finally, comparison between metastases and primaries suggests methylation patterns are established early and maintained through disease progression for both ER+ and ER- tumors. Bisulfite-converted DNA from 397 lesions were analyzed with the Illumina GoldenGate Methylation Cancer Panel I array.
Project description:We generated prostatic stromal microarray expression data for functional validation of our LOH/AI hot-/cold-spots in stroma. Stromal cells from normal peripheral zone tissue and from tumors are grown in culture, and were analyed on gene expression platform. Stromal cells cultured from normal peripheral zone tissues (F-PZ-64, F-PZ-79, F-PZ-82, F-PZ-102, F-PZ-105) and from tumors (F-CA-31, F-CA-39, F-CA-52, F-CA-67, F-CA-93) were established and grown as previously described in Peehl et al. (2000). Total RNAs were extracted from semi-confluent cells (passages 4-5) one day after feeding fresh medium using RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions and treated with TURBO DNA-free kit (Ambion). Hybridizations were performed according to Illumina protocols by the Genomic Core, Lerner Research Institute, Cleveland Clinic. All samples were hybridized to the Illumina Sentrix Human-6_v3 Expression BeadChip (GPL6884) which contains 48,000 distinct oligonucleotide probes. Normalization is done using average normalization algorithm of BeadStudio Gene Expression Module v3.4 (Illumina).
Project description:The purpose of this study was to develop a comprehensive picture of the STZ-diabetic host transcriptional response during the acute stage of melioidosis and investigate the interplay between the diabetic host response and Burkholderia pseudomallei. To adress this, we compared the transcriptome of infected STZ-diabetic liver and spleen with uninfected STZ-diabetic tissues over an infection period of 42 hr (16 hpi, 24 hpi and 42 hpi, respectively) to identify genes whose expression is altered in response to an acute infection. The microarray experimental design included 3 biological replicates for each time point and both liver and spleen were harvested for RNA extraction. The microarray data from each time point was compared relative to uninfected diabetic mice. In addition, the transcriptome of uninfected STZ-diabetic liver and spleen were also compared with uninfected buffer (sodium citrate) control mice.
Project description:Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes are genomic modification events that occur in germinal center (GC) B cells and are initiated through deamination of cytidine to uracil by activation induced cytidine deaminase (AID). Resulting uracil-guanine (U-G) mismatches are subsequently processed by low-fidelity base-excision (BER) and mismatch repair (MMR) pathways to yield mutations and DNA strand lesions. Although off-target AID activity also contributes to oncogenic point mutations and chromosome translocations associated with B cell lymphomas, the role of downstream AID-associated DNA repair pathways in the pathogenesis of these lymphomas is unknown. Here, we show that simultaneous BER and MMR deficiency causes genomic instability and a shorter latency to the development of a BCL6-driven GC B cell lymphoma. In contrast, loss of BER alone is highly protective against B cell transformation while loss of MMR fosters the development of a variety of malignancies. These findings lend insight into a complex interplay between AID-associated BER and MMR pathways that produces a net protective effect against GC B cell lymphomagenesis. Representative B cell lymphomas from 3 IµHABcl6, 6 IµHABcl6 Ung-/- Msh2-/-, and 5 IµHABcl6 Msh2-/- mice were analyzed in this study. Total RNA was extracted from frozen tumor cells and processed according to Illumina standard protocols.
Project description:Human CD34+ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34+ cells to cytokines caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34+ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols. 3 replicates of control unstimulated (Group 1), cytokine stimulated, cytokine strimulated and treated with polybren and cytokine stimulated and transduced with lentiviral vector coding for GFP in the presence of polybren were analysed