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: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: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: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:The B-cell receptor (BCR) plays an important role in pathogenesis and progression of chronic lymphocytic leukemia (CLL). We investigated the BCR triggering-dependent microRNA modulation by stimulating CLL cells with immobilized anti-IgM. miRome of immobilized anti-IgM stimulated CLL cells (n=16) identified a substantial upregulation of miR-132 in both unmutated (UM) and mutated (M) IGHV subgroups. A parallel gene expression profile and an in-silico analysis to identify miR-132 target genes¸ allowed us to focus on SIRT1, that encodes for a histone deacetylase targeting several proteins including TP53. We defined a reduction of SIRT1 protein levels upon immobilized anti-IgM stimulation (P=0.001), and a concomitant increase in TP53 acetylation (P=0.0072). The TP53 target gene CDKN1A was consistently up-regulated in immobilized anti-IgM stimulated CLL cells. Of note, the miR-132 constitutive expression levels in CLL cases (n=134) were of similar magnitude of those obtained in in vitro immobilized anti-IgM stimulated CLL cells. Additionally, high miR-132 expression levels retained a favorable prognostic impact in M (P=0.005), but not in UM CLL patients (P=0.968). The described miR-132/SIRT1/TP53 axis, sequentially characterized by BCR triggering, miR-132 up-regulation, SIRT1 down-regulation and TP53 acetylation, should be considered in the light of emerging drugs targeting the BCR pathway in CLL. investigated the BCR triggering-dependent gene expression modulation by stimulating CLL cells with immobilized anti-IgM.
Project description:The B-cell receptor (BCR) plays an important role in pathogenesis and progression of chronic lymphocytic leukemia (CLL). We investigated the BCR triggering-dependent mRNA modulation by stimulating CLL cells with immobilized anti-IgM. miRome of immobilized anti-IgM stimulated CLL cells (n=16) identified a substantial upregulation of miR-132 in both unmutated (UM) and mutated (M) IGHV subgroups. A parallel gene expression profile and an in-silico analysis to identify miR-132 target genes¸ allowed us to focus on SIRT1, that encodes for a histone deacetylase targeting several proteins including TP53. We defined a reduction of SIRT1 protein levels upon immobilized anti-IgM stimulation (P=0.001), and a concomitant increase in TP53 acetylation (P=0.0072). The TP53 target gene CDKN1A was consistently up-regulated in immobilized anti-IgM stimulated CLL cells. Of note, the miR-132 constitutive expression levels in CLL cases (n=134) were of similar magnitude of those obtained in in vitro immobilized anti-IgM stimulated CLL cells. Additionally, high miR-132 expression levels retained a favorable prognostic impact in M (P=0.005), but not in UM CLL patients (P=0.968). The described miR-132/SIRT1/TP53 axis, sequentially characterized by BCR triggering, miR-132 up-regulation, SIRT1 down-regulation and TP53 acetylation, should be considered in the light of emerging drugs targeting the BCR pathway in CLL. Investigated the BCR triggering-dependent gene expression modulation by stimulating CLL cells with immobilized anti-IgM.