Project description:miR-223 is step-wise increasingly up-regulated in the normal esophagus - Barrett's esophagus -esophageal adenocarcinoma carcinoma sequence. In this study, we aimed to determine the function of miR-223 in esophageal adenocarcinoma carcinogenesis.
Project description:miR-223 is step-wise increasingly up-regulated in the normal esophagus - Barrett's esophagus -esophageal adenocarcinoma carcinoma sequence. In this study, we aimed to determine the function of miR-223 in esophageal adenocarcinoma carcinogenesis. miR-223 was transfected in OE33 cells using 10nM pre-miR hsa-miR-223 miRNA precursor (Ambion, Life Technologies, Grand Island, NY) and lipofectamin 2000 (OE33_223_1 and OE33_223_2). Mock control OE33 cells were transfected with a negative control pre-miR miRNA (OE33_NEG_1 and OE33_NEG_2). HumanHT-12 v4 Expression BeadChip arrays (Illumina, San Diego, CA) were used for microarray hybridizations to examine the global gene expression of two biological replicated experiments (four samples in total). The array targets more than 25,000 annotated genes with 47,323 unique probes derived from the National Center for Biotechnology Information (NCBI) Reference Sequence (RefSeq) Release 38 and UniGene (Build 199) databases.
Project description:Identifying biomarkers predictive for early esophageal cancer detection is critical considering the dismal survival rates. We investigated the involvement of microRNAs (miRNAs), their utility as biomarkers, and their association with survival in esophageal cancer, including Barrett’s associated and sporadic adenocarcinoma (ADC), and squamous cell carcinoma (SCC). MiRNA expression was measured in cancerous and adjacent non-cancerous tissue pairs collected from 76 US and Japanese patients enrolled in 3 distinct cohorts. In ADC patients, miR-21, miR-194, miR-293, and miR-223 expression was elevated, while miR-375 and miR-203 expression was reduced in cancerous tissue compared to non-cancerous tissue. Increased levels of miR-192 and miR-194 were observed in Barrett’s associated compared to sporadic ADC cancerous tissue. In SCC patients, miR-21, miR-181b, miR-155, and miR-146b expression was elevated while miR-375 and miR-203 levels were reduced in cancerous tissue compared to non-cancerous tissue. Significantly, elevated mir-21 expression in non-cancerous tissue was strongly associated with worse prognosis, independent of nodal status and age. Sample classification using miRNA expression yielded accuracies as high as 86% for diagnosis and 78% for Barrett’s esophagus status. Our results highlight that miRNAs are deregulated in esophageal carcinogenesis and Barrett’s esophagus, and that their expression is associated with survival in cancer patients. Sample classification using miRNA expression demonstrates their potential utility as biomarkers for esophageal carcinoma diagnosis.
Project description:Identifying biomarkers predictive for early esophageal cancer detection is critical considering the dismal survival rates. We investigated the involvement of microRNAs (miRNAs), their utility as biomarkers, and their association with survival in esophageal cancer, including Barrett’s associated and sporadic adenocarcinoma (ADC), and squamous cell carcinoma (SCC). MiRNA expression was measured in cancerous and adjacent non-cancerous tissue pairs collected from 76 US and Japanese patients enrolled in 3 distinct cohorts. In ADC patients, miR-21, miR-194, miR-293, and miR-223 expression was elevated, while miR-375 and miR-203 expression was reduced in cancerous tissue compared to non-cancerous tissue. Increased levels of miR-192 and miR-194 were observed in Barrett’s associated compared to sporadic ADC cancerous tissue. In SCC patients, miR-21, miR-181b, miR-155, and miR-146b expression was elevated while miR-375 and miR-203 levels were reduced in cancerous tissue compared to non-cancerous tissue. Significantly, elevated mir-21 expression in non-cancerous tissue was strongly associated with worse prognosis, independent of nodal status and age. Sample classification using miRNA expression yielded accuracies as high as 86% for diagnosis and 78% for Barrett’s esophagus status. Our results highlight that miRNAs are deregulated in esophageal carcinogenesis and Barrett’s esophagus, and that their expression is associated with survival in cancer patients. Sample classification using miRNA expression demonstrates their potential utility as biomarkers for esophageal carcinoma diagnosis. MiRNA microarray expression was measured using miRNA microarray chips version 3 (Ohio State University) in 44 SCC cases and 32 ADC cases, of which 18 were also diagnosed with Barrett’s esophagus.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:41 lung adenocarcinoma from never-smokers hybridized on Illumina SNP arrays on 13 HumanCNV370-Quadv3 chips. High-resolution array comparative genomic hybridization analysis of lung adenocarcinoma in 41 never smokers for identification of new minimal common regions (MCR) of gain or loss. The SNP array analysis validated copy-number aberrations and revealed that RB1 and WRN were altered by recurrent copy-neutral loss of heterozygosity.The present study has uncovered new aberrations containing cancer genes. The oncogene FUS is a candidate gene in the 16p region that is frequently gained in never smokers. Multiple genetic pathways defined by gains of MYC, deletions of RB1 and WRN or gains on 7p and 7q are involved in lung adenocarcinoma in never smokers. A 'Cartes d'Identite des Tumeurs' (CIT) project from the French National League Against Cancer (http://cit.ligue-cancer.net) 41 samples hybridized on Illumina SNP arrays. Submitter : Fabien PETEL petelf@ligue-cancer.net . Project leader : Pr Pierre FOURET pierre.fouret@psl.aphp.fr
Project description:We used a multi-omics approach combining transcriptomics, proteomics and metabolomics to study the impact of over-expression and inhibition of the microRNA miR-223, a pleiotropic regulator of metabolic-related disease, in the RAW monocyte-macrophage cell line. We analyzed the levels of proteins, mRNAs, and metabolites in order to identify genes involved in miR-223 regulation, to determine candidate disease biomarkers and potential therapeutic targets. We observed that both up- and down-regulation of miR-223 induced profound changes in the mRNA, protein and metabolite profiles in RAW cells. Microarray-based transcriptomics evidenced a change in 120 genes that were linked predominantly to histone acetylation, bone remodeling and RNA regulation. In addition, 30 out the 120 genes encoded long noncoding RNAs. The nanoLC-MS/MS revealed that 52 proteins were significantly altered when comparing scramble, pre- and anti-miR-223 treatments. Sixteen out of the mRNAs coding these proteins genes are predicted to have binding sites for miR-223. CARM-1, Ube2g2, Cactin and Ndufaf4 were confirmed to be miR-223 targets by western blotting. Analyses using Gene Ontology annotations evidenced association with cell death, splicing and stability of mRNAs, bone remodeling and cell metabolism. miR-223 alteration changed the expression of CARM-1, Ube2g2, Cactin and Ndufaf4 during osteoclastogenesis and macrophage, indicating that these genes are potential biomarkers of these processes. The most important discriminant metabolites found in the metabolomics study were found to be hydrophilic amino acids, carboxylic acids linked to metabolism and pyrimidine nucleotides, indicating that changes in miR-223 expression alter the metabolic profile of cells, and may affect their apoptotic and proliferative state.