Lentivirus-mediated antagomir expression for specific inhibition of miRNA function

Micro RNAs (miRNA) regulate gene expression by hybridization and recruitment of multi-protein complexes to complementary mRNA target sequences. miRNA function can transiently be antagonized by antagomirs—chemically modified oligonucleotides complementary to individual miRNAs. Here, we describe the induction of stable loss-of-function phenotypes for specific miRNAs by lentivirus-mediated antagomir expression. Lentivirally expressed antagomirs are transcribed from a H1-promoter located within the lentiviral 3′LTR and were directed against miRNAs encoded on the polycistronic miR17-92 transcript. Functional silencing of miR-18a, miR-19b and miR-20a by the corresponding antagomirs specifically relieves miRNA-mediated reporter gene repression. Inhibition of miRNA function correlates to reduction of ‘miRNA’ amplification by miRNA-specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 antagomirs in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals individual and complementary functions of miR-18a and miR-20a and demonstrates specific miRNA impact on cell proliferation in a cell culture model

NK-like homeodomain proteins activate NOTCH3-signaling in leukemic T-cells

<p>Abstract</p> <p>Background</p> <p>Homeodomain proteins control fundamental cellular processes in development and in cancer if deregulated. Three members of the NK-like subfamily of homeobox genes (NKLs), TLX1, TLX3 and NKX2-5, are implicated in T-cell acute lymphoblastic leukemia (T-ALL). They are activated by particular chromosomal aberrations. However, their precise function in leukemogenesis is still unclear. Here we screened further NKLs in 24 T-ALL cell lines and identified the common expression of MSX2. The subsequent aim of this study was to analyze the role of MSX2 in T-cell differentiation which may be disturbed by oncogenic NKLs.</p> <p>Methods</p> <p>Specific gene activity was examined by quantitative real-time PCR, and globally by expression profiling. Proteins were analyzed by western blot, immuno-cytology and immuno-precipitation. For overexpression studies cell lines were transduced by lentiviruses.</p> <p>Results</p> <p>Quantification of MSX2 mRNA in primary hematopoietic cells demonstrated higher levels in CD34+ stem cells as compared to peripheral blood cells and mature CD3+ T-cells. Furthermore, analysis of MSX2 expression levels in T-cell lines after treatment with core thymic factors confirmed their involvement in regulation. These results indicated that MSX2 represents an hematopoietic NKL family member which is downregulated during T-cell development and may functionally substituted by oncogenic NKLs. For functional analysis JURKAT cells were lentivirally transduced, overexpressing either MSX2 or oncogenic TLX1 and NKX2-5, respectively. These cells displayed transcriptional activation of NOTCH3-signaling, including NOTCH3 and HEY1 as analyzed by gene expression profiling and quantitative RT-PCR, and consistently attenuated sensitivity to gamma-secretase inhibitor as analyzed by MTT-assays. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with NOTCH-pathway repressors, SPEN/MINT/SHARP and TLE1/GRG1, representing a potential mechanism for (de)regulation. Finally, elevated expression of NOTCH3 and HEY1 was detected in primary TLX1/3 positive T-ALL cells corresponding to the cell line data.</p> <p>Conclusion</p> <p>Identification and analysis of MSX2 in hematopoietic cells implicates a modulatory role via NOTCH3-signaling in early T-cell differentiation. Our data suggest that reduction of NOTCH3-signaling by physiological downregulation of MSX2 expression during T-cell development is abrogated by ectopic expression of oncogenic NKLs, substituting MSX2 function.</p

Neuraminidase-1 promotes heart failure after ischemia/reperfusion injury by affecting cardiomyocytes and invading monocytes/macrophages

Neuraminidase (NEU)1 forms a multienzyme complex with beta-galactosidase (β-GAL) and protective-protein/cathepsin (PPC) A, which cleaves sialic-acids from cell surface glycoconjugates. We investigated the role of NEU1 in the myocardium after ischemia/reperfusion (I/R). Three days after inducing I/R, left ventricles (LV) of male mice (3 months-old) displayed upregulated neuraminidase activity and increased NEU1, β-GAL and PPCA expression. Mice hypomorphic for neu1 (hNEU1) had less neuraminidase activity, fewer pro-inflammatory (Li

miR-125b regulates chemotaxis and survival of bone marrow derived granulocytes in vitro and in vivo.

The evolutionary conserved miR-125b is highly expressed in hematopoietic stem cells (HSC) enhancing self-renewal and survival. Accordingly, over-expression of miR-125b in HSC may induce myeloproliferative neoplasms and leukemia with long latency. During hematopoietic cell maturation miR-125b expression decreases, and the function of miR-125b in mature granulocytes is not yet known. We here use transplantation of miR-125b over-expressing HSC into syngeneic hosts to generate and analyse miR-125b over-expressing granulocytes. Under steady state conditions, miR-125b over-expression inhibits granulocytic chemotaxis and LPS- but not PMA- and TNFα- induced cell death. Inflammatory signals modulate the effects of miR-125b over-expression as demonstrated in a sterile peritonitis and a polymicrobial sepsis model. In particular, survival of mice with miR-125b over-expressing granulocytes is significantly reduced as compared to controls in the polymicrobial sepsis model. These data demonstrate inflammation dependent effects of miR-125b in granulocytes and may point to therapeutic intervention strategies in the future

BCR-ABL affects STAT5A and STAT5B differentially.

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors linking extracellular signals to target gene transcription. Hematopoietic cells express two highly conserved STAT5-isoforms (STAT5A/STAT5B), and STAT5 is directly activated by JAK2 downstream of several cytokine receptors and the oncogenic BCR-ABL tyrosine kinase. Using an IL-3-dependent cell line with inducible BCR-ABL-expression we compared STAT5-activation by IL-3 and BCR-ABL in a STAT5-isoform specific manner. RNAi targeting of STAT5B strongly inhibits BCR-ABL-dependent cell proliferation, and STAT5B but not STAT5A is essential for BCL-XL-expression in the presence of BCR-ABL. Although BCR-ABL induces STAT5-tyrosine phosphorylation independent of JAK2-kinase activity, BCR-ABL is less efficient in inducing active STAT5A:STAT5B-heterodimerization than IL-3, leaving constitutive STAT5A and STAT5B-homodimerization unaffected. In comparison to IL-3, nuclear accumulation of a STAT5A-eGFP fusion protein is reduced by BCR-ABL, and BCR-ABL tyrosine kinase activity induces STAT5A-eGFP translocation to the cell membrane and co-localization with the IL-3 receptor. Furthermore, BCR-ABL-dependent phosphorylation of Y682 in STAT5A was detected by mass-spectrometry. Finally, RNAi targeting STAT5B but not STAT5A sensitizes human BCR-ABL-positive cell lines to imatinib-treatment. These data demonstrate differences between IL-3 and BCR-ABL-mediated STAT5-activation and isoform-specific effects, indicating therapeutic options for isoform-specific STAT5-inhibition in BCR-ABL-positive leukemia

NKL homeobox gene activities in hematopoietic stem cells, T-cell development and T-cell leukemia

<div><p>T-cell acute lymphoblastic leukemia (T-ALL) cells represent developmentally arrested T-cell progenitors, subsets of which aberrantly express homeobox genes of the NKL subclass, including TLX1, TLX3, NKX2-1, NKX2-5, NKX3-1 and MSX1. Here, we analyzed the transcriptional landscape of all 48 members of the NKL homeobox gene subclass in CD34+ hematopoietic stem and progenitor cells (HSPCs) and during lymphopoiesis, identifying activities of nine particular genes. Four of these were expressed in HSPCs (HHEX, HLX1, NKX2-3 and NKX3-1) and three in common lymphoid progenitors (HHEX, HLX1 and MSX1). Interestingly, our data indicated downregulation of NKL homeobox gene transcripts in late progenitors and mature T-cells, a phenomenon which might explain the oncogenic impact of this group of genes in T-ALL. Using MSX1-expressing T-ALL cell lines as models, we showed that HHEX activates while HLX1, NKX2-3 and NKX3-1 repress MSX1 transcription, demonstrating the mutual regulation and differential activities of these homeobox genes. Analysis of a public T-ALL expression profiling data set comprising 117 patient samples identified 20 aberrantly activated members of the NKL subclass, extending the number of known NKL homeobox oncogene candidates. While 7/20 genes were also active during hematopoiesis, the remaining 13 showed ectopic expression. Finally, comparative analyses of T-ALL patient and cell line profiling data of NKL-positive and NKL-negative samples indicated absence of shared target genes but instead highlighted deregulation of apoptosis as common oncogenic effect. Taken together, we present a comprehensive survey of NKL homeobox genes in early hematopoiesis, T-cell development and T-ALL, showing that these genes generate an NKL-code for the diverse stages of lymphoid development which might be fundamental for regular differentiation.</p></div

Analyses of NKL homeobox genes in T-ALL cell lines.

<p>(A) RQ-PCR analysis of HHEX after siRNA-mediated knockdown in LOUCY. Concomitant reduction of transcript levels indicated that LYL1 (left) and GATA3 (middle) activate the expression of HHEX. Forced expression of TAL1 resulted in slightly increased expression of HHEX (right), indicating an activating input as well. (B) RQ-PCR analysis of HHEX after forced expression of NKL homeobox genes HLX1, NKX2-3 and NKX3-1 in LOUCY (left). RQ-PCR analysis of NKX3-1 after siRNA-mediated knockdown of HHEX in LOUCY (middle). RQ-PCR analysis of NKX3-1 after forced expression of NKL homeobox genes HHEX, HLX1 and NKX2-3 in JURKAT (right). (C) RQ-PCR analysis of BMPR1A and BMPR2 after forced expression of NKL homeobox gene NKX2-3 in LOUCY.</p