Project description:FLASH, a novel method, was utilized to identify binding sites of RNA-binding proteins (RBPs) with single-nucleotide resolution (Nucleic Acids Res 2020;48:e15). In order to identify HuR target genes in THP-1 macrophages after circARCN1 regulation, we performed FLASH experiments using the anti-HuR antibody on THP-1 macrophages with circARCN1 knockdown or overexpression. The resulting FLASH products were then subjected to RNA sequencing.
Project description:We report the HuR-RNA interactions in the liver by performing RNA-immunoprecipitation sequencing (RIP-seq). RIP-seq was performed in healthy livers of wildtype (WT) mice using a HuR antibody. We found that 1380 cytoplasmic-target mRNAs bound to HuR, as assessed by the comparison between the HuR-specific antibody and the IgG control
Project description:Lipopolysaccharide exposure to macrophages induces an inflammatory response that is heavily regulated at the post-transcriptional level. HuR, ELAVL1, is an RNA binding protein that binds and regulate the maturation and half-life of AU/U rich elements (ARE) containing cytokines and chemokines transcripts, mediating the LPS induced response. Here we investigated to what extent small molecules inhibiting HuR-RNA interaction, called tanshinone mimics, counteract the LPS induced macrophage response. We show that tanshinone mimics are present in solution in a keto-enolic tautomerism and that, by molecular dynamic calculations, the ortho quinone form is the preferred species interacting with HuR and favoring the closure of its conformation to a no-binding mode. The protection of the enolic status with diacetate caused the loss of activity of tanshinone mimics in vitro but active in vivo. Murine macrophages cell line RAW264.7 was treated with LPS and tanshinone mimics and the modulation of the LPS induced response was monitored by RNA and Ribonucleoprotein immunoprecipitation sequencing. Correlation analyses indicated that LPS induced a strong coupling between differentially expressed genes and HuR-bound genes and that tanshinone mimics reduced the interaction. Functional annotation addressed a specific set of genes, as Cxcl10, Il1b, Cd40, Fas, involved in chemotaxis and immune response whose association with HuR decreased and led to a reduction of their protein level and secretion. The same effect was observed in primary murine bone marrow derived macrophages and in vivo in a LPS induced peritonitis model, in which the serum level of Cxcl10 and Il1b was strongly reduced, endowing tanshinone mimics with anti-inflammatory properties in vivo.
Project description:Purpose: Tumor hypoxia is a major cause of treatment resistance, especially to radiation therapy at conventional dose rate (CONV), and we wanted to assess whether hypoxia does alter tumor sensitivity to FLASH. Methods and materials: We engrafted several tumor types (glioblastoma [GBM], head and neck cancer, and lung adenocarcinoma) subcutaneously in mice to provide a reliable and rigorous way to modulate oxygen supply via vascular clamping or carbogen breathing. We irradiated tumors using a single 20-Gy fraction at either CONV or FLASH, measured oxygen tension, monitored tumor growth, and sampled tumors for bulk RNAseq and pimonidazole analysis. Next, we inhibited glycolysis with trametinib in GBM tumors to enhance FLASH efficacy. Results: Using various subcutaneous tumor models, and in contrast to CONV, FLASH retained antitumor efficacy under acute hypoxia. These findings show that in addition to normal tissue sparing, FLASH could overcome hypoxia-mediated tumor resistance. Follow-up molecular analysis using RNAseq profiling uncovered a FLASH-specific profile in human GBM that involved cell-cycle arrest, decreased ribosomal biogenesis, and a switch from oxidative phosphorylation to glycolysis. Glycolysis inhibition by trametinib enhanced FLASH efficacy in both normal and clamped conditions. Conclusions: These data provide new and specific insights showing the efficacy of FLASH in a radiation-resistant context, proving an additional benefit of FLASH over CONV.
Project description:Here we profile nascent transcription, RNA polymerase III occupancy, chromatin accessibility, and H3K27ac levels in THP-1 monocytes and THP-1 derived macrophages after 72 hr exposure to phorbol myristate acetate (PMA).
Project description:Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability In this dataset, we employed two distinct experiments. 1) HuR RIP-chip to identify mRNA targets of HuR. 2) HuR knockdown to identify mRNAs whose expression are dependent on HuR. All 12 samples were normalized with PLIER using Affymetrix power tools. To identify RNA targets of HuR, HuR RIP samples were compared to Mock RIP samples. To identify RNA regulated by HuR, HuR knockdown samples were compared to mock knockdown samples.
Project description:Comprehensive meta-analysis and target screening confirmed that the mRNA-binding protein of ELAV-family HuR is oncogenic and universally upregulated in brain tumors, which highlight HuR as an universal chemotherapeutic target. HuR functionality in cancer cells is strictly dependent on HuR nuclear/cytoplasmic shuttling and dimerization; therefore, we developed a new class of inhibitors of HuR protein dimerization by utilizing medicinal chemistry techniques and reporter cell-based assay of HuR dimerization. The therapeutic potentials of lead compound (SRI-42127) were evaluated in five primary patient-derived glioma xenolines of classic, proneural, and mesenchymal subtypes, in vitro, and in mouse glioma model, in vivo. The Illumina global RNA-Sequencing was performed on PDGx-derived glioma neurospheres of different subtypes after treatment with DMSO (control) or SRI-42127 (3 uM) for 12 h to analysis transcripts and cell-signaling pathways affected by new inhibitor of HuR dimerization.
Project description:Integrative regulatory mapping indicates that the RNA-binding protein HuR (ELAVL1) couples pre-mRNA processing and mRNA stability In this dataset, we employed two distinct experiments. 1) HuR RIP-chip to identify mRNA targets of HuR. 2) HuR knockdown to identify mRNAs whose expression are dependent on HuR.
Project description:HuR-deficient cells showed the decreased expression of genes involved in chemotaxis, cell proliferation and signal transduction. We used microarray to detail the global gene expression of HuR-deficient cell after poly(I:C) stimulation.