Silencing of Bruton's tyrosine kinase (Btk) using short interfering RNA duplexes (siRNA)

Heinonen, Juhana E.; Smith, C. I. Edvard; Nore, Beston F.

doi:10.1016/s0014-5793(02)03206-4

Cited by 49 publications

(47 citation statements)

References 28 publications

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“…The database consists of 30 studies and contains 398 siRNAs targeting 92 genes. For the analysis only siRNAs of length 19 (duplex region) were used (287 siRNAs-17 studies, 30 genes) [6,[18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33]. Expression levels are either at the RNA or protein level.…”

Section: Methodsmentioning

confidence: 99%

Improved and automated prediction of effective siRNA

Chalk

Wahlestedt

Sonnhammer

2004

Biochemical and Biophysical Research Communications

119

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Short interfering RNAs are used in functional genomics studies to knockdown a single gene in a reversible manner. The results of siRNA experiments are highly dependent on the choice of siRNA sequence. In order to evaluate siRNA design rules, we collected a database of 398 siRNAs of known efficacy from 92 genes. We used this database to evaluate previously proposed rules from smaller datasets, and to find a new set of rules that are optimal for the entire database. We also trained a regression tree with full crossvalidation. It was however difficult to obtain the same precision as methods previously tested on small datasets from one or two genes. We show that those methods are overfitting as they work poorly on independent validation datasets from multiple genes. Our new design rules can predict siRNAs with efficacy P50% in 91% of cases, and with efficacy P90% in 52% of cases, which is more than a twofold improvement over random selection. Software for designing siRNAs is available online via a web server at http:// sisearch.cgb.ki.se/ or as a standalone version for high-throughput applications. Ó 2004 Elsevier Inc. All rights reserved.RNAi is a recently discovered biological phenomenon whereby a single gene may be inhibited at the RNA stage of synthesis. short interfering RNAs (siRNAs) are duplexes of two RNA molecules, typically 21-mers with a 2nt 3 0 overhang [1]. One strand is loaded into the RISC complex [2] after which a sequence specific cleavage of the target takes place. The strength of the principle behind any form of RNA targeting (siRNA and antisense) is that the molecule can be used to inhibit expression of any mRNA, and thus the protein it encodes. This effect can be demonstrated without affecting related proteins, making it an invaluable tool for functional genomics. siRNAs have been found to be effective in Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, and mammals [3]. Many reviews of the biological processes behind siRNA inhibition exist, see for example [3,4].Efficient reliable design of high efficacy siRNA molecules is essential to meet the needs for cost-effective high-throughput functional genomics projects. To meet these needs the siRNAs designed should at least conform to the following criteria: (a) be predicted with high accuracy, (b) be sequence specific, and (c) be produced in a form that facilitates high-throughput production. In this paper we address these criteria, focusing primarily on a and b.Despite the apparent ease of designing siRNAs (compared to a popular DNA-based knockdown technique: antisense oligonucleotides (AOs)), a number of problems still remain. Randomly selected siRNAs produce knockdown P50% with 58-78% success rate, while very effective siRNAs ( P90/95%) are found by chance 11-18% of the time [5,6].Initial design paradigms for siRNAs were based on motif rules, such as AAN (19)

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Section: Methodsmentioning

confidence: 99%

Improved and automated prediction of effective siRNA

Chalk

Wahlestedt

Sonnhammer

2004

Biochemical and Biophysical Research Communications

119

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“…Two different target sequences, one localized in the SH2 domain (B#1 shRNA) of Btk, the other localized in the SH3 domain (B#2 shRNA) of Btk, were selected. 23 U937-TD cells were transduced with constructs containing these oligonucleotides or the vector control (VC) resulting in the U937-TD-B#1, U937-TD-B#2 and U937-TD-VC cell lines, respectively. Figure 2A shows that the B#1 shRNA efficiently reduced Btk expression in U937-TD cells, leading to approximately 96% down-regulation of Btk expression relative to the vector control cells.…”

Section: Btk Is a Positive Regulator Of Trem-1 Signaling And Functionmentioning

confidence: 99%

Btk is a positive regulator in the TREM-1/DAP12 signaling pathway

Ormsby

Schlecker

Ferdin

et al. 2011

Blood

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IntroductionTREM-1 belongs to the immunoglobulin (Ig)-like superfamily of receptors. In humans, TREM-1 is expressed on neutrophils and CD14 high monocytes. 1 TREM-1 expression is further up-regulated by TLR ligands. Binding of TREM-1 to endogenous ligand(s) expressed on granulocytes and platelets and present in sera of septic patients, 2-4 as well as to exogenous ligands on Marburg and Ebola viruses, 5 has been described. The exact nature of TREM-1 ligand(s), however, remains elusive. Engagement of TREM-1 by agonistic monoclonal antibodies (mAbs) results in respiratory burst, degranulation, phagocytosis, secretion of pro-inflammatory cytokines such as TNF-␣, of the chemokines, IL-8 and monocyte chemotactic protein-1 (MCP-1), and in the up-regulation of cell surface expressed differentiation/activation markers. 1 In animal models of lipopolysacharide (LPS)-induced septic shock and microbial sepsis caused by live Escherichia coli, application of a soluble TREM-1-Ig fusion protein greatly increased survival of experimental animals indicating the importance of TREM-1 in the amplification of inflammation. 6 TREM-1 possesses a short intracellular part that lacks intrinsic signaling motifs. Instead, it is coupled to the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, DAP12. 1 TREM-1 engagement leads to Ca 2ϩ mobilization and phosphorylation of several proteins including DAP12, extracellular-signal regulated kinase (Erk1/2), phospholipase C␥ (PLC␥) 1 and the adaptor protein NTAL that further interacts with Grb2. 7 Recently, it was reported that the adaptor protein CARD9 is essential for TREM-1-induced secretion of TNF-␣, IL-2 and IL-12p40 by mouse bone marrow-derived dendritic cells (BMDCs). 8 Bruton tyrosine kinase (Btk), a member of the Tec family of protein tyrosine kinases (PTKs), is involved in signaling via a variety of receptors including the B-cell receptor (BCR), cytokine receptors and integrins. 9,10 Loss of Btk function causes X-linked agammaglobulinemia (XLA), a rare primary immunodeficiency disease. 11,12 Mutations causing XLA have been described in all Btk domains as well as in noncoding sequences of the gene. 13 XLA is manifested by severe defects in early B-cell development, resulting in an almost complete absence of peripheral B cells and Igs of all classes. Affected individuals suffer from recurrent bacterial and enteroviral infections. 9 In mice, the R28C point mutation in the pleckstrin homology (PH) domain of Btk leads to X-linked immunodeficiency (Xid). 14 In innate immune cells, the role of Btk is less clear. Btk has been implicated in several pathways in myeloid cells. 10 Btk associates with certain TLRs and their downstream signaling molecules. 15 Contradictory results were obtained from studies of TLR stimulation of monocytes of XLA patients. In one report, relative to healthy controls, monocytes of XLA patients secreted reduced amounts of TNF-␣ upon LPS stimulation. 16 On the other hand, Perez et al 17 described that monocytes of XLA patients showed similar TN...

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“…0 -GACAAAAACGUGUCUCGCCTT-3 0 ) using siPORT NeoFX Transfection Agent (Ambion) as described earlier. 33 The transfected cells were used for subsequent experiments 48 h later.…”

Section: Immunoprecipitation and Western Blot Analysismentioning

confidence: 99%

Inhibition of autophagy induced by overexpression of mda-7/interleukin-24 strongly augments the antileukemia activity in vitro and in vivo

Yang

Yin

et al. 2009

Cancer Gene Ther

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Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a novel candidate of tumor suppressor that can selectively induce apoptosis experimentally in a spectrum of human cancer cells including leukemia cells. However, a recent study suggests that mda-7/IL-24 promotes the survival of chronic lymphocytic leukemia B-cells. In this study, we showed that mda-7/IL-24 was constitutively expressed in leukemia cell lines and primary acute myeloid leukemia samples. Using a conditionally replicating adenovirus expressing mda-7/IL-24 (ZD55-IL-24), we showed that enforced expression of mda-7/IL-24 in leukemia cells induced autophagy, which was triggered by the upregulation of Beclin-1. Immunofluorescence and coimmunoprecipitation studies suggested that mda-7/IL-24 protein interacts with Beclin-1. Class III PI3K/Beclin-1 complex was shown involved in the mda-7/IL-24-induced autophagy. Moreover, autophagy inhibition by phosphatidylinositol 3-kinase inhibitor, wortmannin, resulted in a reduced Beclin-1 expression and autophagosome formation associated with significantly enhanced cell death. Importantly, the combination of ZD55-IL-24 with wortmannin elicited a strongly enhanced antileukemia efficacy in established leukemia xenografts. These results suggest that mda-7/IL-24-induced autophagy in leukemia cells may provide survival advantage and mda-7/IL-24 combined with agents that disrupt autophagy is a promising new strategy for the treatment of leukemia.

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Silencing of Bruton's tyrosine kinase (Btk) using short interfering RNA duplexes (siRNA)

Cited by 49 publications

References 28 publications

Improved and automated prediction of effective siRNA

Improved and automated prediction of effective siRNA

Btk is a positive regulator in the TREM-1/DAP12 signaling pathway

Inhibition of autophagy induced by overexpression of mda-7/interleukin-24 strongly augments the antileukemia activity in vitro and in vivo

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