A Ten-microRNA Expression Signature Predicts Survival in Glioblastoma

Glioblastoma (GBM) is the most common and aggressive primary brain tumor with very poor patient median survival. To identify a microRNA (miRNA) expression signature that can predict GBM patient survival, we analyzed the miRNA expression data of GBM patients (n = 222) derived from The Cancer Genome Atlas (TCGA) dataset. We divided the patients randomly into training and testing sets with equal number in each group. We identified 10 significant miRNAs using Cox regression analysis on the training set and formulated a risk score based on the expression signature of these miRNAs that segregated the patients into high and low risk groups with significantly different survival times (hazard ratio [HR] = 2.4; 95% CI = 1.4–3.8; p<0.0001). Of these 10 miRNAs, 7 were found to be risky miRNAs and 3 were found to be protective. This signature was independently validated in the testing set (HR = 1.7; 95% CI = 1.1–2.8; p = 0.002). GBM patients with high risk scores had overall poor survival compared to the patients with low risk scores. Overall survival among the entire patient set was 35.0% at 2 years, 21.5% at 3 years, 18.5% at 4 years and 11.8% at 5 years in the low risk group, versus 11.0%, 5.5%, 0.0 and 0.0% respectively in the high risk group (HR = 2.0; 95% CI = 1.4–2.8; p<0.0001). Cox multivariate analysis with patient age as a covariate on the entire patient set identified risk score based on the 10 miRNA expression signature to be an independent predictor of patient survival (HR = 1.120; 95% CI = 1.04–1.20; p = 0.003). Thus we have identified a miRNA expression signature that can predict GBM patient survival. These findings may have implications in the understanding of gliomagenesis, development of targeted therapy and selection of high risk cancer patients for adjuvant therapy

The role of HuR in the post-transcriptional regulation of interleukin-3 in T cells.

Human Interleukin-3 (IL-3) is a lymphokine member of a class of transiently expressed mRNAs harboring Adenosine/Uridine-Rich Elements (ARE) in their 3' untranslated regions (3'-UTRs). The regulatory effects of AREs are often mediated by specific ARE-binding proteins (ARE-BPs). In this report, we show that the human IL-3 3'-UTR plays a post-transcriptional regulation role in two human transformed cell lines. More specifically, we demonstrate that the hIL-3 3'-UTR represses the translation of a luciferase reporter both in HeLa and Jurkat T-cells. These results also revealed that the hIL-3 3'-UTR-mediated translational repression is exerted by an 83 nt region comprised mainly by AREs and some non-ARE sequences. Moreover, electrophoretic mobility shift assays (EMSAs) and UV-crosslinking analysis show that this hIL-3 ARE-rich region recruits five specific protein complexes, including the ARE-BPs HuR and TIA-1. HuR binding to this ARE-rich region appears to be spatially modulated during T-cell activation. Together, these results suggest that HuR recognizes the ARE-rich region and plays a role in the IL-3 3'-UTR-mediated post-transcriptional control in T-cells

Self-assembled organic–inorganic magnetic hybrid adsorbent ferrite based on cyclodextrin nanoparticles

Organic–inorganic magnetic hybrid materials (MHMs) combine a nonmagnetic and a magnetic component by means of electrostatic interactions or covalent bonds, and notable features can be achieved. Herein, we describe an application of a self-assembled material based on ferrite associated with β-cyclodextrin (Fe-Ni/Zn/βCD) at the nanoscale level. This MHM and pure ferrite (Fe-Ni/Zn) were used as an adsorbent system for Cr3+ and Cr2O72− ions in aqueous solutions. Prior to the adsorption studies, both ferrites were characterized in order to determine the particle size distribution, morphology and available binding sites on the surface of the materials. Microscopy analysis demonstrated that both ferrites present two different size domains, at the micro- and nanoscale level, with the latter being able to self-assemble into larger particles. Fe-Ni/Zn/βCD presented smaller particles and a more homogeneous particle size distribution. Higher porosity for this MHM compared to Fe-Ni/Zn was observed by Brunauer–Emmett–Teller isotherms and positron-annihilation-lifetime spectroscopy. Based on the pKa values, potentiometric titrations demonstrated the presence of βCD in the inorganic matrix, indicating that the lamellar structures verified by transmission electronic microscopy can be associated with βCD assembled structures. Colloidal stability was inferred as a function of time at different pH values, indicating the sedimentation rate as a function of pH. Zeta potential measurements identified an amphoteric behavior for the Fe-Ni/Zn/βCD, suggesting its better capability to remove ions (cations and anions) from aqueous solutions compared to that of Fe-Ni/Zn

RNA-binding proteins ranging from 34-88 kDa recognize the hIL-3 ARE-rich region.

<p>(A) IL-3 Up, ARE and Down radiolabeled RNAs were incubated with HeLa cytoplasmic protein extracts (lanes 2–4) and the RNA-protein interactions were UV cross-linked. Samples were subjected to RNase digestion and to SDS-10% polyacrylamide gel electrophoresis. (B) UV-cross linking assays were also performed with Jurkat cytoplasmic cell extracts. IL-3 Up, ARE and Down radiolabeled RNAs were incubated with Jurkat cytoplasmic protein extracts (lanes 6-8).</p

T cell activation modulates HuR binding towards the hIL-3 ARE-rich region.

<p>(A) HuR EMSA supershift analysis was carried out with Jurkat cytoplasmic extracts activated at 0, 6, 12 and 24 hours. (B) Graphic representation of HuR supershift quantification during T cell activation. Values represent mean ± standard error of the mean (SEM) from two experiments. Fold changes were normalized to 0 hours of T cell-activation with PMA/Ionomycin.</p

The hIL-3 ARE-rich region is recognized by specific RNA-binding protein complexes in both HeLa and Jurkat T-cells.

<p>(A) Radiolabeled RNA probes corresponding to the hIL-3 3'-UTR UP, ARE and Down regions were incubated with (lanes 5–8) or without (lanes 1-4) cytoplasmic extracts from HeLa cells. Arrows indicate the obtained gel shifts. (B) To assess the specificity of the RBP complexes that recognize the IL-3 ARE-rich region in HeLa cells, an EMSA competition assay was performed using cold RNA competitors at increasing fold-excess (10¹–10⁴): Up (lanes 2–5), ARE (lanes 6–9) or Down (lanes 10–13). Non- competitor RNA (nc) was added in lane 1. The pGem7z multiple cloning site (80nt) was used as a negative control RNA (c-RNA). (C) EMSAs were also performed using cytoplasmic extracts from Jurkat cells. (D) EMSA reactions were treated with 15 μg of Proteinase K (PK).</p

The hIL-3 ARE-rich region is recognized by HuR and TIA-1 ARE-BPs.

<p>(A) The ³²P-labeled IL-3 ARE-rich sequence was incubated with HeLa cytoplasmic protein extracts and TIA-1, TIAR, AUF-1 and HuR antibodies. Non-immune goat serum (IgG) antibody was used as a negative control (lane 7). IL-3 ARE incubated with the HeLa cytoplasmic extract without antibody addition was used as an additional control in the analysis (lane 1). (B) Jurkat cytoplasmic protein extracts were also used in the EMSA super shift assays.</p