MicroRNAs (miRNA) comprise a group of short ribonucleic acid molecules implicated in regulation of key biological processes and functions at the post-transcriptional level. Ionizing radiation (IR) causes DNA damage and generally triggers cellular stress response. However, the role of miRNAs in IR-induced response in human embryonic stem cells (hESC) has not been defined yet. Here, by using system biology approaches, we show for the first time, that miRNAome undergoes global alterations in hESC ( ...[more]
Project description:Cytoplasmic RNA bound to eIF4E was pulled down from MDA-MB-231 cells to determine the influence of radiation on eIF4E mRNA binding 4 samples were analyzed with 3 biological replicates
Project description:Inactivation of Pten occurs by multiple mechanisms including epigenetic silencing, point mutations, insertion, and deletion, which are tissue dependent. Although frequent loss of heterozygosity around Pten locus and plausible involvement of epigenetic silencing have been reported in radiation-induced thymic lymphomas, the frequency of Pten inactivation and the spectrum of causal aberrations have not yet been extensively characterized. Here, we assessed the principal mode of inactivation by comprehensively analyzing expression and alterations of Pten gene in 23 radiation-induced thymic lymphomas developed in B6C3F1 mice. We found no evidence for methylation-associated silencing of Pten gene. Instead, we found complex structural abnormalities in 8 lymphomas (35%) that included missense and nonsense mutations, 1- and 3-bp insertions, and focal deletions. Sequencing of deletion breakpoints suggested that illegitimate V(D)J recombination and microhomology-mediated rearrangement were responsible for the focal deletions. Seven out of these 8 lymphomas had biallelic alterations, and 4 of them did not express any Pten protein. These aberrations of Pten were well coincided with downstream Akt phosphorylation on Ser473. In conclusion, Pten inactivation, which is frequently biallelic and is caused by a variety of structural abnormalities but not by epigenetic silencing, is involved in radiation-induced lymphomagenesis. Three thymic lymphomas were analyzed by array-CGH method.
Project description:Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment. Murine progenitor cells, JB6 cell line, were stably transformed with Annexin A2 shRNA or vector only control and then treated with ionizing radiation at two doses, 10cGy and 100cGy. Samples were collected at 2hr, 6hr and 24hr for RNA isolation. Two untreated time points were collected also: 2hr and 24hr.
Project description:Human pancreatic islets were isolated from pancreas of deceased donors by Ricordi's procedure and cultured in CMRL 1066 medium additioned with human albumin. EVs were isolated from conditioned medium derived from islet culture after isolation. Once isolated, RNA of islets and islet-derived EVs was extracted and analyzed for microRNA expression within 48 hours after isolation.
Project description:MOLT4 cells in log phase (1x106 cells/ml) were irradiated with 4 Gy at room temperature, at a dose rate of 2.45 Gy/minute with a 137Cs ?-irradiator. After 4 hours, actinomycin D (5 ?M) was added to block transcription. Aliquots of culture were removed at hourly intervals and RNA was extracted (Trizol; Invitrogen). RNA and cRNA quantity and quality was determined by Nanodrop spectrophotometer and Bioanalyser 2100 (Agilent). Transcript levels were determined at different time points using Affymetrix Human U133A microarrays.<br><br>
Project description:MOLT4 cells in log phase (1x106 cells/ml) were pre-treated for 2 hours with the I?B? phosphorylation inhibitor BAY 11-7082 (Calbiochem) at 5?M, and the control samples with corresponding volume of DMSO. Cells were then irradiated with 4 Gy at room temperature, at a dose rate of 2.45 Gy/minute with a 137Cs ?-irradiator. Cells were harvested 2 hours after irradiation and RNA was extracted (Trizol; Invitrogen). RNA and cRNA quantity and quality was determined by Nanodrop spectrophotometer and Bioanalyser 2100 (Agilent). Affymetrix GeneChip® Human Genome U133A 2.0 arrays were hybridized as standard (www.affymetrix.co.uk). Array quality was determined using R and Affymetrix Expression Console file values. We then calculated a Z-score which represents the degree to which BAY 11-7082 reduces irradiation-induced transcription of all target genes.