Project description:To obtain a PTC cell model, primary human thyrocytes have been infected with a retrovirus expressing RET/PTC1 oncogene, using parental thyrocytes as control. The obtained RET/PTC-dependent differential miRNA expression profile, representing the effects of RET/PTC1 oncogene present in about one third of papillary thyroid carcinoma (PTC), models the early event of thyrocytes transformation ending to PTC. miRNA expression profiles of thyrocytes expressing RET/PTC1 oncogene and parental thyrocytes were compared. Biological replicates could not be generated.
Project description:To obtain a PTC cell model, primary human thyrocytes have been infected with a retrovirus expressing RET/PTC1 oncogene, using parental thyrocytes as control. The obtained RET/PTC-dependent differential miRNA expression profile, representing the effects of RET/PTC1 oncogene present in about one third of papillary thyroid carcinoma (PTC), models the early event of thyrocytes transformation ending to PTC.
Project description:Mass populations of thyrocytes stably expressing wild type RET/PTC1 oncogene or RET/PTC1 carrying Y451F mutation and parental thyrocytes were used for hybridization on Affymetrix HG-U133A and HG-U133B chips. For each cell condition were generated two different targets (indicated as two different samples in the database, i.e. "Parental Thyrocytes" and "Parental Thyrocytes bis")for a total number of six samples. For the data analysis the two samples from the same condition (i.e. Parental thyrocytes) were considered as duplicates.
Project description:Genomic rearrangements leading to intragenic gene fusion are mainly found in some types of haematopoietic malignancies and sarcomas. Recently they have been described also in carcinomas such as the papillary thyroid histotype (60%-70%) and the Hürthle thyroid tumours (58%). The presence of junction oncogene constitutes an area of exciting research for emerging therapy as targeting the RET-PTC1 fusion oncogene by using small interfering RNA (siRNA) strategies since it is present only in the tumour cells and not in the surrounding normal cells. Therefore, we developed a siRNA against RET-PTC1 junction and assess its efficiency on the human papillary thyroid carcinoma cell line TPC-1 which spontaneously harbours the RET-PTC1 oncogene. The targeted genes are assessed by microarray analysis by comparing the regulated genes by the siRNA_RET-PTC1 vs a siRNA_RET developed on the RET part of the mRNA minus the siRNA_control that contain four mutation within the RET-PTC1 sequence. To test the targeted genes in the TPC-1 cell line that spontaneously harbours RET-PTC1 junction of two siRNAs developed: Within the RET-PTC1 junction, and in the mRNA RET part. A non-specific siRNA harbouring 4 mutations within the RET-PTC1 sequence was used as negative control (siRNA_control). By real-time PCR (Q-RT-PCR) we demonstrated that both siRNAs (siRNA_RET-PTC1 and siRNA_RET) significantly reduce RET mRNA levels of about 85 % in TPC-1 cells. The negative control did not show an effect of RET mRNA levels. Three independent transfections were performed on TPC-1 cells using 5 µl of Lipofectamine 2000 transfection reagent (Invitrogen, Cergy-Pontoise, France) and 50nM of i) siRNA_RET-PTC1 or ii) siRNA_RET or iii) siRNA_control that harbour 4 mutations within its sequence. Total RNAs of untreated cells and transfected cells were purified using the RNA cleanup and concentration kit (QIAGEN, Hilden, Germany) and gathered in 4 pools : 1) TPC-1 harbouring RET-PTC1 ; 2) TPC-1 silenced for RET-PTC1 with siRNA RET-PTC1 ; 3) TPC-1 silenced for siRNA RET ; 4) TPC-1 treated with the siRNA control.
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes
Project description:We have sequenced miRNA libraries from human embryonic, neural and foetal mesenchymal stem cells. We report that the majority of miRNA genes encode mature isomers that vary in size by one or more bases at the 3’ and/or 5’ end of the miRNA. Northern blotting for individual miRNAs showed that the proportions of isomiRs expressed by a single miRNA gene often differ between cell and tissue types. IsomiRs were readily co-immunoprecipitated with Argonaute proteins in vivo and were active in luciferase assays, indicating that they are functional. Bioinformatics analysis predicts substantial differences in targeting between miRNAs with minor 5’ differences and in support of this we report that a 5’ isomiR-9-1 gained the ability to inhibit the expression of DNMT3B and NCAM2 but lost the ability to inhibit CDH1 in vitro. This result was confirmed by the use of isomiR-specific sponges. Our analysis of the miRGator database indicates that a small percentage of human miRNA genes express isomiRs as the dominant transcript in certain cell types and analysis of miRBase shows that 5’ isomiRs have replaced canonical miRNAs many times during evolution. This strongly indicates that isomiRs are of functional importance and have contributed to the evolution of miRNA genes Sequence library of miRNAs from a single sample of human foetal mesenchymal stem cells. Results tested and confirmed by northern blotting. Please note that only raw data files are available for the embryonic and neual samples and thus, directly submitted to SRA (SRX547311, SRX548700, respectively under SRP042115/PRJNA247767)
Project description:Gene expression profiling of immortalized human mesenchymal stem cells with hTERT/E6/E7 transfected MSCs. hTERT may change gene expression in MSCs. Goal was to determine the gene expressions of immortalized MSCs.
Project description:Transcriptional profiling of human mesenchymal stem cells comparing normoxic MSCs cells with hypoxic MSCs cells. Hypoxia may inhibit senescence of MSCs during expansion. Goal was to determine the effects of hypoxia on global MSCs gene expression.
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:Single-nucleus RNA sequencing (snRNA-seq) was used to profile the transcriptome of 16,015 nuclei in human adult testis. This dataset includes five samples from two different individuals. This dataset is part of a larger evolutionary study of adult testis at the single-nucleus level (97,521 single-nuclei in total) across mammals including 10 representatives of the three main mammalian lineages: human, chimpanzee, bonobo, gorilla, gibbon, rhesus macaque, marmoset, mouse (placental mammals); grey short-tailed opossum (marsupials); and platypus (egg-laying monotremes). Corresponding data were generated for a bird (red junglefowl, the progenitor of domestic chicken), to be used as an evolutionary outgroup.