PDF - 1331K, Western Blot Analysis.

PDF - 42K, miRNAs implicated in several cardiovascular events were also differentially expressed following fractionated irradiation in HCAEC.

Supplementary Table S2 from NS-398, ibuprofen, and cyclooxygenase-2 RNA interference produce significantly different gene expression profiles in prostate cancer cells

PDF - 56K, Microarray data depicting gene expression fold changes respective to control group in genes from stress response category following SD and MF radiation exposure at 6h and 24h.

PDF - 64K, Supplementary Materials and Methods

PDF - 34K, Changes in selected differentially expressed genes identified by microarray analysis were confirmed by real-time RT-PCR.

PDF - 22K, Genes differentially expressed (> 2-fold change, P < 0.05) in response to SD and MF irradiations were classified into functional categories by gene ontology classification. Enrichment of individual functional gene categories was determined by hypergeometric distribution of P values obtained from comparison of the number of the number of genes differentially expressed to the number of annotated genes in each category.

PDF - 61K, Legends for Supplementary Tables

PDF - 46K, Microarray data depicting gene expression fold changes respective to control group in genes from immune response category following SD and MF radiation exposure at 6h and 24h.

PDF - 29K, Differentially expressed Target mRNAs and regulatory miRNAs (showing inverse correlation) from cardiovascular pathways in HCAEC at 24h following SD and MF irradiation were identified by Target Filter Analysis program.

There are no existing treatments for the long-term degenerative effects of traumatic brain injury (TBI). This is due, in part, to our limited understanding of chronic TBI and uncertainty about which proposed mechanisms for long-term neurodegeneration are amenable to treatment with existing or novel drugs. Here, we used microarray and pathway analyses to interrogate TBI-induced gene expression in the rat hippocampus and cortex at several acute, subchronic and chronic intervals (24 hours, 2 weeks, 1, 2, 3, 6 and 12 months) after parasagittal fluid percussion injury. We used Ingenuity pathway analysis (IPA) and Gene Ontology enrichment analysis to identify significantly expressed genes and prominent cell signaling pathways that are dysregulated weeks to months after TBI and potentially amenable to therapeutic modulation. We noted long-term, coordinated changes in expression of genes belonging to canonical pathways associated with the innate immune response (i.e., NF-κB signaling, NFAT signaling, Complement System, Acute Phase Response, Toll-like receptor signaling, and Neuroinflammatory signaling). Bioinformatic analysis suggested that dysregulation of these immune mediators-many are key hub genes-would compromise multiple cell signaling pathways essential for homeostatic brain function, particularly those involved in cell survival and neuroplasticity. Importantly, the temporal profile of beneficial and maladaptive immunoregulatory genes in the weeks to months after the initial TBI suggests wider therapeutic windows than previously indicated.

Patients with traumatic brain injury (TBI) are frequently diagnosed with depression. Together, these two leading causes of death and disability significantly contribute to the global burden of healthcare costs. However, there are no drug treatments for TBI and antidepressants are considered off-label for depression in patients with TBI. In molecular profiling studies of rat hippocampus after experimental TBI, we found that TBI altered the expression of a subset of small, non-coding, microRNAs (miRNAs). One known neuroprotective compound (17β-estradiol, E2), and two experimental neuroprotective compounds (JM6 and PMI-006), reversed the effects of TBI on miRNAs. Subsequent in silico analyses revealed that the injury-altered miRNAs were predicted to regulate genes involved in depression. Thus, we hypothesized that drug-induced miRNA profiles can be used to identify compounds with antidepressant properties. To confirm this hypothesis, we examined miRNA expression in hippocampi of injured rats treated with one of three known antidepressants (imipramine, fluoxetine and sertraline). Bioinformatic analyses revealed that TBI, potentially via its effects on multiple regulatory miRNAs, dysregulated transcriptional networks involved in neuroplasticity, neurogenesis, and circadian rhythms- networks known to adversely affect mood, cognition and memory. As did E2, JM6, and PMI-006, all three antidepressants reversed the effects of TBI on multiple injury-altered miRNAs. Furthermore, JM6 reduced TBI-induced inflammation in the hippocampus and depression-like behavior in the forced swim test; these are both properties of classic antidepressant drugs. Our results support the hypothesis that miRNA expression signatures can identify neuroprotective and antidepressant properties of novel compounds and that there is substantial overlap between neuroprotection and antidepressant properties.

Introducción: el cáncer gástrico es muy frecuente en Colombia y es la primera causa de muerte por cáncer. De acuerdo con la cascada de Correa existen unas etapas progresivas en la formación de esta enfermedad, que van de la gastritis, pasando por la atrofia, metaplasia y displasia, hasta el cáncer. En esas etapas intermedias se pudiera detectar y prevenir, pero no existen marcadores en sangre diferentes al pepsinógeno que puedan ayudar a detectar las etapas premalignas ni a diagnosticar el cáncer. Por lo cual, las investigaciones que ayuden a descubrir nuevos biomarcadores son de gran importancia.Objetivos: el objetivo de este trabajo es identificar marcadores moleculares (perfil de expresión de ARNm) que distingan a los pacientes con condiciones premalignas (atrofia, metaplasia) y cáncer gástrico, de aquellos pacientes que solo tienen gastritis. Metodología: se tomaron pacientes en cada una de las etapas de la cascada de Correa, los cuales proporcionaron una muestra, previo consentimiento firmado, de 2,5 mL de sangre en ayunas para el análisis de expresión de genes tomadas después de la endoscopia inicial. La sangre se colocó en un tubo de ARN sanguíneo PAXgene; luego, el ARN se extrajo de la sangre y se analizó usando una plataforma de microarrays, los cuales identificaron cambios de expresión de ARN mensajero que permitieron diferenciar cada una de las etapas descritas.Resultados: se incluyeron 89 pacientes, y en los hallazgos endoscópicos se encontró gastritis crónica antral en 27 pacientes (30,3%), cáncer gástrico avanzado en 25 (28%), pangastritis crónica en 15 (16,8%), cáncer temprano en 7 (7,8%), sospecha de metaplasia intestinal en 6 (6,7%), sospecha de gastritis atrófica en 3 (3,3%) y úlcera péptica en 2 casos (2,2%). Cuando se revisó el informe patológico se halló gastritis crónica en 34 pacientes (22 mujeres), adenocarcinoma intestinal en 20 (4 mujeres), metaplasia intestinal 18 casos (13 mujeres), cáncer tipo difuso en 11 (7 mujeres), displasia de bajo grado en 4 y de alto grado en 1, y atrofia sola en 1 paciente. En el análisis de expresión genética se encontraron 48 genes que permitieron determinar a los pacientes con gastritis crónica de aquellos con cáncer gástrico. También se hallaron 14 genes para diferenciar los pacientes con cáncer difuso de los de tipo intestinal, y un grupo de 48 genes que ayudó a distinguir los pacientes con gastritis crónica de los de metaplasia intestinal.Conclusiones: este es el primer trabajo en Colombia, y a nivel mundial, que permite identificar nuevos biomarcadores a través de la expresión genética del ARN mensajero, el cual diferencia en sangre las etapas de la cascada de Correa, y permite diagnosticar el cáncer gástrico. Es probable que en un futuro se puedan utilizar como una prueba diagnóstica o de seguimiento.

Virally mediated RNA interference (RNAi) to knock down injury-induced genes could improve functional outcome after traumatic brain injury (TBI); however, little is known about the consequences of gene knockdown on downstream cell signaling pathways and how RNAi influences neurodegeneration and behavior. Here, we assessed the effects of adeno-associated virus (AAV) siRNA vectors that target two genes with opposing roles in TBI pathogenesis: the allegedly detrimental neuronal nitric oxide synthase (nNOS) and the potentially protective glutathione peroxidase 1 (GPx-1). In rat hippocampal progenitor cells, three siRNAs that target different regions of each gene (nNOS, GPx-1) effectively knocked down gene expression. However, in vivo, in our rat model of fluid percussion brain injury, the consequences of AAV-siRNA were variable. One nNOS siRNA vector significantly reduced the number of degenerating hippocampal neurons and showed a tendency to improve working memory. GPx-1 siRNA treatment did not alter TBI-induced neurodegeneration or working memory deficits. Nevertheless, microarray analysis of laser captured, virus-infected neurons showed that knockdown of nNOS or GPx-1 was specific and had broad effects on downstream genes. Since nNOS knockdown only modestly ameliorated TBI-induced working memory deficits, despite widespread genomic changes, manipulating expression levels of single genes may not be sufficient to alter functional outcome after TBI.

The underlying molecular mechanisms of how dysregulated microRNAs (miRNAs) cause neurodegeneration after traumatic brain injury (TBI) remain elusive. Here we analyzed the biological roles of approximately 600 genes - we previously found these dysregulated in dying and surviving rat hippocampal neurons - that are targeted by ten TBI-altered miRNAs. Bioinformatic analysis suggests that neurodegeneration results from a global miRNA-mediated suppression of genes essential for maintaining proteostasis; many are hub genes - involved in RNA processing, cytoskeletal metabolism, intracellular trafficking, cell cycle progression, repair/maintenance, bioenergetics and cell-cell signaling - whose disrupted expression is linked to human disease. Notably, dysregulation of these essential genes would significantly impair synaptic function and functional brain connectivity. In surviving neurons, upregulated miRNA target genes are co-regulated members of prosurvival pathways associated with cellular regeneration, neural plasticity, and development. This study captures the diversity of miRNA-regulated genes that may be essential for cell repair and survival responses after TBI.

Background and aims: Adjuvant oxaliplatin-based chemotherapy is widely used for stage III colorectal cancer (CRC) after curative surgery.CRC is a molecularly heterogeneous disease, and our current knowledge of therapeutic response-related genetic factors remains limited.GALNT14-rs9679162 genotype is a prognostic predictor for chemotherapy response in advanced hepatocellular carcinoma.Here, we investigated whether this genotype was related to the therapeutic outcome of stage III CRC. Materials and Methods: A cohort of 300 stage III CRC patients receiving curative resection followed by oxaliplatin-based chemotherapy was retrospectively recruited.GALNT14 genotypes and the clinicopathological factors were correlated with post-therapeutic prognosis.Results: Of these patients, 18% patients had GALNT14-rs9679162 "TT" and 82% had the "GT" + "GG" genotypes.The analysis showed that the "TT" genotype was associated with unfavorable overall survival (OS, P = 0.009) but not with recurrence free survival (RFS, P = 0.700).Subgroup analysis showed that the "TT" genotype was associated with unfavorable OS in the following subgroups: age £ 65 years, male, left side CRC, N2 stage, CEA > 5 ng/ml, and mucinous histology (P = 0.012, 0.011, 0.009, 0.025, 0.013, and 0.007, respectively).Within the latter 2 subgroups, the "TT" genotype was the only independent predictor for OS.Finally, the "TT" genotype was associated with the T4 tumor stage (P = 0.017) and in patients with T4 tumors, the "TT" genotype was the only independent predictor for unfavorable RFS (P = 0.007).Conclusions: GALNT14 "TT" genotype was associated with unfavorable OS in stage III CRC patients receiving curative surgery and adjuvant oxaliplatin-based chemotherapy.

Abstract Although modern radiotherapy technologies can precisely deliver higher doses of radiation to tumors, thus, reducing overall radiation exposure to normal tissues, moderate dose, and normal tissue toxicity still remains a significant limitation. The present study profiled the global effects on transcript and miR expression in human coronary artery endothelial cells using single-dose irradiation (SD, 10 Gy) or multifractionated irradiation (MF, 2 Gy × 5) regimens. Longitudinal time points were collected after an SD or final dose of MF irradiation for analysis using Agilent Human Gene Expression and miRNA microarray platforms. Compared with SD, the exposure to MF resulted in robust transcript and miR expression changes in terms of the number and magnitude. For data analysis, statistically significant mRNAs (2-fold) and miRs (1.5-fold) were processed by Ingenuity Pathway Analysis to uncover miRs associated with target transcripts from several cellular pathways after irradiation. Interestingly, MF radiation induced a cohort of mRNAs and miRs that coordinate the induction of immune response pathway under tight regulation. In addition, mRNAs and miRs associated with DNA replication, recombination and repair, apoptosis, cardiovascular events, and angiogenesis were revealed. Implications: Radiation-induced alterations in stress and immune response genes in endothelial cells contribute to changes in normal tissue and tumor microenvironment, and affect the outcome of radiotherapy. Mol Cancer Res; 12(7); 1002–15. ©2014 AACR.

We assessed changes in cell lines of varying p53 status after various fractionation regimens to determine if p53 influences gene expression and if multifractionated (MF) irradiation can induce molecular pathway changes. LNCaP (p53 wild-type), PC3 (p53 null), and DU145 (p53 mutant) prostate carcinoma cells received 5 and 10 Gy as single-dose (SD) or MF (0.5 Gy x 10, 1 Gy x 10, and 2 Gy x 5) irradiation to simulate hypofractionated and conventionally fractionated prostate radiotherapies, respectively. mRNA analysis revealed 978 LNCaP genes differentially expressed (greater than two-fold change, P < .05) after irradiation. Most were altered with SD (69%) and downregulated (75%). Fewer PC3 (343) and DU145 (116) genes were induced, with most upregulated (87%, 89%) and altered with MF irradiation. Gene ontology revealed immune response and interferon genes most prominently expressed after irradiation in PC3 and DU145. Cell cycle regulatory (P = 9.23 x 10(-73), 14.2% of altered genes, nearly universally downregulated) and DNA replication/repair (P = 6.86 x 10(-30)) genes were most prominent in LNCaP. Stress response and proliferation genes were altered in all cell lines. p53-activated genes were only induced in LNCaP. Differences in gene expression exist between cell lines and after varying irradiation regimens that are p53 dependent. As the duration of changes is ≥24 hours, it may be possible to use radiation-inducible targeted therapy to enhance the efficacy of molecular targeted agents.

We have previously demonstrated that prostate carcinoma cells exposed to fractionated radiation differentially expressed more genes compared to single-dose radiation. To understand the role of miRNA in regulation of radiation-induced gene expression, we analyzed miRNA expression in LNCaP, PC3 and DU145 prostate cancer cells treated with single-dose radiation and fractionated radiation by microarray. Selected miRNAs were studied in RWPE-1 normal prostate epithelial cells by RT-PCR. Fractionated radiation significantly altered more miRNAs as compared to single-dose radiation. Downregulation of oncomiR-17-92 cluster was observed only in the p53 positive LNCaP and RWPE-1 cells treated with single-dose radiation and fractionated radiation. Comparison of miRNA and mRNA data by IPA target filter analysis revealed an inverse correlation between miR-17-92 cluster and several targets including TP53INP1 in p53 signaling pathway. The base level expressions of these miRNAs were significantly different among the cell lines and did not predict the radiation outcome. Tumor suppressor miR-34a and let-7 miRNAs were upregulated by fractionated radiation in radiosensitive LNCaP (p53 positive) and PC3 (p53-null) cells indicating that radiation-induced miRNA expression may not be regulated by p53 alone. Our data support the potential for using fractionated radiation to induce molecular targets and radiation-induced miRNAs may have a significant role in predicting radiosensitivity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have come under scrutiny because of the gastrointestinal, renal, and cardiovascular toxicity associated with prolonged use of these drugs. The purpose of this study was to identify molecular targets for NSAIDs related to cellular toxicity with a view to optimize drug efficacy in the clinic. Coronary artery smooth muscle cells and endothelial cells were treated with low (clinically achievable) and high (typically used in preclinical studies) concentrations of celecoxib, NS398, and ibuprofen for 24 hours. NSAIDs-induced gene expression changes were evaluated by microarray analysis and validated by real-time reverse-transcription polymerase chain reaction and western blotting. The functional significance of differentially expressed genes was evaluated by Ingenuity Pathway Analysis. At high concentrations, NSAIDs altered the expression of genes regulating cell proliferation and cell death. NSAIDs also altered genes associated with cardiovascular functions including inflammation, thrombosis, fibrinolysis, coronary artery disease, and hypertension. The gene expression was most impacted by ibuprofen, celecoxib, and NS398, in that order. This study revealed that NSAIDs altered expression of an array of genes associated with cardiovascular events and emphasizes the potential for fingerprinting drugs in preclinical studies to assess the potential drug toxicity and to optimize the drug efficacy in clinical settings.