Home About us Contact
|
Home About us Contact | ||
|
|
||
Short Hairpin RNA (short + hairpin_rna)
Selected AbstractsSuppression of the mouse double minute 4 gene causes changes in cell cycle control in a human mesothelial cell line responsive to ultraviolet radiation exposureENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 9 2009Melisa Bunderson-Schelvan Abstract The TP53 tumor suppressor gene is the most frequently inactivated gene in human cancer identified to date. However, TP53 mutations are rare in human mesotheliomas, as well as in many other types of cancer, suggesting that aberrant TP53 function may be due to alterations in its regulatory pathways. Mouse double minute 4 (MDM4) has been shown to be a key regulator of TP53 activity, both independently as well as in concert with its structural homolog, Mouse Double Minute 2 (MDM2). The purpose of this study was to characterize the effects of MDM4 suppression on TP53 and other proteins involved in cell cycle control before and after ultraviolet (UV) exposure in MeT5a cells, a nonmalignant human mesothelial line. Short hairpin RNA (shRNA) was used to investigate the impact of MDM4 on TP53 function and cellular transcription. Suppression of MDM4 was confirmed by Western blot. MDM4 suppressed cells were analyzed for cell cycle changes with and without exposure to UV. Changes in cell growth as well as differences in the regulation of direct transcriptional targets of TP53, CDKN1A (cyclin-dependent kinase 1,, p21) and BAX, suggest a shift from cell cycle arrest to apoptosis upon increasing UV exposure. These results demonstrate the importance of MDM4in cell cycle regulation as well as a possible role inthe pathogenesis of mesothelioma-type cancers. Environ. Mol. Mutagen. 2009. © 2009 Wiley-Liss, Inc. [source] RNA interference by expressing short hairpin RNA in the Ciona intestinalis embryoDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2008Aya Nishiyama We carried out RNA interference by expressing short hairpin RNA (shRNA) in the Ciona intestinalis embryo. For this purpose, we identified a gene encoding U6 small nuclear RNA (snRNA) in the C. intestinalis genome. The 1-kb sequence upstream of the U6 snRNA gene was sufficient for directing transcription of short RNA as revealed by Northern blot hybridization. An shRNA-expressing plasmid vector was constructed, in which shRNA-encoding oligonucleotides are inserted downstream of the U6 promoter. An shRNA that contained a sequence homologous to the C. intestinalis tyrosinase gene (Ci-tyrosinase) suppressed melanization of pigment cells in the brain of morphologically normal tailbud embryos. An shRNA that perfectly matched the translated sequence of enhanced green fluorescent protein (EGFP) (a mutant type of Aequorea victoria green fluorescent protein) suppressed the expression of the coelectroporated EGFP transgene. These results suggest that the expression of shRNA interferes with functions of both endogenous and exogenous genes. The shRNA-expressing plasmid constructed in the present study provides an easy and inexpensive alternative for the functional analysis of genes in ascidian embryos. [source] Essential role of C/EBP, in G-CSF-induced transcriptional activation and chromatin modification of myeloid-specific genesGENES TO CELLS, Issue 4 2008Satoshi Iida Granulocyte colony-stimulating factor (G-CSF) regulates the proliferation and differentiation of neutrophilic progenitor cells. Here, we investigated the roles of CCAAT/enhancer-binding protein (C/EBP), in the G-CSF-induced transcriptional activation and chromatin modification of the CCR2 and myeloperoxidase (MPO) genes in IL-3-dependent myeloid FDN1.1 cells. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays revealed that G-CSF activates C/EBP, to bind target promoters. ChIP mapping experiments across the CCR2 and MPO genes showed that G-CSF induces histone H3 modifications: the acetylation of Lys9, trimethylation of Lys4 and trimethylation of Lys9. The distribution profile of the trimethylated Lys9 was distinct from that of the two other modifications. All the G-CSF-induced C/EBP, recruitment, transcriptional activation and histone modifications were reversed by re-stimulation with IL-3, and were abolished by short hairpin RNA (shRNA)-mediated knockdown of C/EBP,. These results indicate that C/EBP, is activated by G-CSF to bind target promoters, and plays critical roles in the transcriptional activation and dynamic chromatin modification of target genes during neutrophil differentiation. [source] Oxidized low-density lipoprotein induces matrix metalloproteinase-9 expression via a p42/p44 and JNK-dependent AP-1 pathway in brain astrocytesGLIA, Issue 1 2009Hui-Hsin Wang Abstract Upregulation of matrix metalloproteinases (MMPs), especially MMP-9, by oxidized low-density lipoprotein (oxLDL) is implicated in many inflammatory diseases including brain injury. However, the signaling mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes largely remain unknown. Here we report that oxLDL induces expression of proMMP-9 via a MAPK-dependent AP-1 activation in rat brain astrocyte (RBA)-1 cells. Results revealed by gelatin zymography, RT-PCR, and Western blotting analyses showed that oxLDL-induced proMMP-9 gene expression was mediated through Akt, JNK1/2, and p42/p44 MAPK phosphorylation in RBA-1 cells. These responses were attenuated by inhibitors of PI3K (LY294002), JNK (SP600125), and p42/p44 MAPK (PD98059), or transfection with dominant negative mutants and short hairpin RNA. Moreover, we demonstrated that AP-1 (i.e., c-Fos/c-Jun) is crucial for oxLDL-induced proMMP-9 expression which was attenuated by pretreatment with AP-1 inhibitor (curcumin). The regulation of MMP-9 gene transcription by AP-1 was confirmed by oxLDL-stimulated MMP-9 luciferase activity which was totally lost in cells transfected with the AP-1 binding site-mutated MMP-9 promoter construct (mt-AP1-MMP-9). These results suggested that oxLDL-induced proMMP-9 expression is mediated through PI3K/Akt, JNK1/2, and p42/p44 MAPK leading to AP-1 activation. Understanding the regulatory mechanisms underlying oxLDL-induced MMP-9 expression in astrocytes might provide a new therapeutic strategy of brain injuries and diseases. © 2008 Wiley-Liss, Inc. [source] Cell cycle effects resulting from inhibition of hepatocyte growth factor and its receptor c-Met in regenerating rat livers by RNA interference,HEPATOLOGY, Issue 6 2007Shirish Paranjpe Hepatocyte growth factor (HGF) and its receptor c-Met are involved in liver regeneration. The role of HGF and c-Met in liver regeneration in rat following two-thirds partial hepatectomy (PHx) was investigated using RNA interference to silence HGF and c-Met in separate experiments. A mixture of 2 c-Met-specific short hairpin RNA (ShRNA) sequences, ShM1 and ShM2, and 3 HGF-specific ShRNA, ShH1, ShH3, and ShH4, were complexed with linear polyethylenimine. Rats were injected with the ShRNA/PEI complex 24 hours before and at the time of PHx. A mismatch and a scrambled ShRNA served as negative controls. ShRNA treatment resulted in suppression of c-Met and HGF mRNA and protein compared with that in controls. The regenerative response was assessed by PCNA, mitotic index, and BrdU labeling. Treatment with the ShHGF mixture resulted in moderate suppression of hepatocyte proliferation. Immunohistochemical analysis revealed severe suppression of incorporation of BrdU and complete absence of mitosis in rats treated with ShMet 24 hours after PHx compared with that in controls. Gene array analyses indicated abnormal expression patterns in many cell-cycle- and apoptosis-related genes. The active form of caspase 3 was seen to increase in ShMet-treated rats. The TUNEL assay indicated a slight increase in apoptosis in ShMet-treated rats compared with that in controls. Conclusion: The data indicated that in vivo silencing of c-Met and HGF mRNA by RNA interference in normal rats results in suppression of mRNA and protein, which had a measurable effect on proliferation kinetics associated with liver regeneration. (HEPATOLOGY 2007.) [source] The role of ZIP8 down-regulation in cadmium-resistant metallothionein-null cellsJOURNAL OF APPLIED TOXICOLOGY, Issue 5 2009Hitomi Fujishiro Abstract The mechanisms of cellular cadmium uptake in mammalian cells remain obscure. To solve this problem, we established cadmium-resistant cells (A7 and B5) from metallothionein-null mouse cells, and found that cadmium accumulation was markedly suppressed in these cells. DNA microarray and real-time PCR analyses revealed that expressions of ZIP (Zrt-, Irt-related protein) 8 and ZIP14 were down-regulated in A7 and B5 cells. In particular, both mRNA and protein levels of ZIP8 were markedly suppressed in A7 and B5 cells. Introduction of short hairpin RNA (shRNA) of ZIP8 into parental cells reduced the accumulation of cadmium to about 35% of that of mock-transfected cells, whereas the introduction of shRNA of divalent metal transporter 1 hardly changed cadmium accumulation. Thus, the cadmium resistance in A7 and B5 cells may be conferred primarily by the down-regulation of ZIP8. In mouse tissues, high expression of ZIP8 was noted in the liver, kidney, lung and testis. These data suggest that ZIP8 plays an important role in cellular uptake of cadmium. Copyright © 2009 John Wiley & Sons, Ltd. [source] RNAi-mediated inhibition of MSP58 decreases tumour growth, migration and invasion in a human glioma cell lineJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 11-12 2009Wei Lin Abstract MSP58, a 58-kD nuclear microspherule protein, is an evolutionarily conserved nuclear protein implicated in the regulation of gene transcription as well as in malignant transformation. An analysis of mRNA expression by real-time PCR revealed that MSP58 was significantly up-regulated in 29% of high-grade glioblastoma tissues as well as in four glioblastoma cell lines. In the present study, we further evaluated the biological functions of MSP58 in U251 glioma cell proliferation, migration, invasion and tumour growth in vivo by specific MSP58 knockdown using short hairpin RNA (shRNA). We found that MSP58 depletion inhibited glioma cell growth, primarily by inducing cell cycle arrest rather than apoptosis. MSP58 depletion also decreased the invasive capability of glioma cells and anchorage-independent colony formation in soft agar. Moreover, suppression of MSP58 expression significantly impaired the growth of glioma xenografts in nude mice. Finally, a cell cycle-associated gene array revealed potential molecular mechanisms contributing to cell cycle arrest in MSP58-depleted glioma cells. In summary, our data highlight the importance of MSP58 in glioma progression and provided a biological basis for MSP58 as a novel candidate target for treatment of glioma. [source] Bmi-1 is critical for the proliferation and invasiveness of gastric carcinoma cellsJOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY, Issue 3 2010Wei Li Abstract Background and Aim:, Bmi-1 is a transcriptional repressor belonging to the Polycomb group and is associated with the cell proliferation and carcinogenesis of a variety of human cancers. The level of Bmi-1 expression correlates with the aggressiveness of many cancers, and is considered an important marker for cancer diagnosis. However, its role in gastric carcinoma is unknown. Methods:, We used lentiviral mediated interfering short hairpin RNA to knockdown Bmi-1 expression in gastric carcinoma human gastric cancer cell line (AGS cells), then tested the cell proliferation by MTT assay, rate of colony formation by colony formation assay, cell cycle distribution by fluorescence-activated cell sorting and cell invasiveness by cell invasion assay. To analyze the expression and localization of Bmi-1 in gastric tumor tissues, we further performed the immunohistochemistry analysis on a gastric cancer tissue array. Results:, We found that knocking down Bmi-1 led to slower cell growth, lesser cell invasiveness, decelerated colony formation, and altered cell cycle progression. In addition, a positive relationship between nuclear expression of Bmi-1 and gastric cancer was observed, suggesting that nucleus localization of Bmi-1 in the cells may be a novel marker of gastric cancer. Conclusions:, Our study highlights critical roles for Bmi-1 in gastric cancer, and suggests that Bmi-1 nuclear localization could be an important marker for the diagnosis of gastric cancer. [source] Stat3 is required for anchorage-independent growth and metastasis but not for mammary tumor development downstream of the ErbB-2 oncogene,MOLECULAR CARCINOGENESIS, Issue 2 2010Isaia Barbieri Abstract The oncogenic transcription factor Stat3 is constitutively active in a high percentage of human tumors including mammary adenocarcinomas and is reported to participate in the ErbB-2 oncogene signaling. In order to assess the role of signal transducer and activator of transcription 3 (Stat3) in mammary tumorigenesis downstream of ErbB-2, we generated mice expressing the activated rat ErbB-2 (neu) but lacking Stat3 in the mammary epithelium. Stat3 is apparently not required for neu-driven mammary tumorigenesis as tumors developed similarly in both Stat3-sufficient and Stat3-deficient glands. However, short hairpin RNA (shRNA)-mediated Stat3 silencing in a neu-overexpressing tumor-derived cell line completely abolished both neu-driven anchorage-independent growth and lung metastasis. Our data suggest that Stat3 might be a useful therapeutic target in breast tumors showing amplification and/or overexpression of the ErbB-2 oncogene, which normally display aggressive, metastatic behavior. © 2009 Wiley-Liss, Inc. [source] Development and characterization of a triple combination gene therapy vector inhibiting HIV-1 multiplicationTHE JOURNAL OF GENE MEDICINE, Issue 10 2008Maria B. Asparuhova Abstract Background RNA-based approaches are promising for long-term gene therapy against HIV-1. They can target virtually any step of the viral replication cycle. It is also possible to combine anti-HIV-1 transgenes targeting different facets of HIV replication to compensate for limitations of any individual construct, maximizing efficacy and decreasing chances of escape mutations. We have previously developed two strategies to inhibit HIV-1 multiplication. One was a short hairpin RNA targeting the host factor cyclophilin A implicated in HIV-1 replication. Additionally, an antisense derivative of U7 small nuclear RNA was designed to induce the skipping of the HIV-1 Tat and Rev internal exons. Results In the present study, we have established an additional tRNAval promoter-driven shRNA against the coding sequence of viral infectivity factor. When human T-cell lines or primary CD4+ T cells are transduced with a triple lentiviral vector encoding these three therapeutic RNAs, HIV-1 multiplication is very efficiently suppressed. Moreover, all three therapeutic RNAs exhibit antiviral effects at early stages of the viral replication cycle (i.e. prior to viral cDNA integration or gene expression). Conclusions These findings make this triple lentiviral vector an attractive candidate for a gene therapy against HIV/AIDS. Copyright © 2008 John Wiley & Sons, Ltd. [source] Lentiviral vectors that carry anti-HIV shRNAs: problems and solutionsTHE JOURNAL OF GENE MEDICINE, Issue 9 2007Olivier ter Brake Abstract Background HIV-1 replication can be inhibited with RNA interference (RNAi) by expression of short hairpin RNA (shRNA) from a lentiviral vector. Because lentiviral vectors are based on HIV-1, viral sequences in the vector system are potential targets for the antiviral shRNAs. Here, we investigated all possible routes by which shRNAs can target the lentiviral vector system. Methods Expression cassettes for validated shRNAs with targets within HIV-1 Leader, Gag-Pol, Tat/Rev and Nef sequences were inserted in the lentiviral vector genome. Third-generation self-inactivating HIV-1-based lentiviral vectors were produced and lentiviral vector capsid production and transduction titer determined. Results RNAi against HIV-1 sequences within the vector backbone results in a reduced transduction titer while capsid production was unaffected. The notable exception is self-targeting of the shRNA encoding sequence, which does not affect transduction titer. This is due to folding of the stable shRNA hairpin structure, which masks the target for the RNAi machinery. Targeting of Gag-Pol mRNA reduces both capsid production and transduction titer, which was improved with a human codon-optimized Gag-Pol construct. When Rev mRNA was targeted, no reduction in capsid production and transduction titer was observed. Conclusions Lentiviral vector titers can be negatively affected when shRNAs against the vector backbone and the Gag-Pol mRNA are expressed during lentiviral vector production. Titer reductions due to targeting of the Gag-Pol mRNA can be avoided with a human codon-optimized Gag-Pol packaging plasmid. The remaining targets in the vector backbone may be modified by point mutations to resist RNAi-mediated degradation during vector production. Copyright © 2007 John Wiley & Sons, Ltd. [source] ORIGINAL ARTICLE: Suppression of Mamu-AG by RNA InterferenceAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 6 2009Jessica G. Drenzek Problem, The role of placental major histocompatibility complex (MHC) class I molecules in pregnancy is not well understood. Mamu-AG, the rhesus monkey homology of human leukocyte antigen (HLA)-G expressed in the human placenta, was targeted for degradation by RNA interference (RNAi), a powerful tool to aid in determining gene function, to determine the effect that this knockdown has on NK cell function. Method of study, A series of potential target short hairpin RNA (shRNA) sequences to suppress Mamu-AG expression was screened, which identified an optimal sequence to use in transfection experiments. Knockdown in two different Mamu-AG-expressing cell lines was measured by flow cytometry. Cytotoxicity assays were performed to correlate Mamu-AG expression with NK cell cytotoxicity. Results, Decreased expression of Mamu-AG by short interfering RNA (siRNA) (70,80%) in cell types tested was associated with increased lysis of Mamu-AG target cells. Conclusion, Target sequences have been identified that knocked down Mamu-AG expression by RNAi and increased lysis by NK cells. This supports the concept that NK cell receptors recognize this placental non-classical MHC class I molecule. [source] Increasing Resistance of Tubular Epithelial Cells to Apoptosis by shRNA Therapy Ameliorates Renal Ischemia-Reperfusion InjuryAMERICAN JOURNAL OF TRANSPLANTATION, Issue 10 2006C. Du Renal tubular epithelial cells (TEC) die by apoptosis or necrosis in renal ischemia-reperfusion injury (IRI). Fas/Fas ligand-dependent fratricide is critical in TEC apoptosis, and Fas promotes renal IRI. Therefore, targeting Fas or caspase-8 may have therapeutic potential for renal injury in kidney transplant or failure. RNA silencing by short hairpin RNA (shRNA) is a novel strategy to down-regulate protein expression. Using this approach, silencing of Fas or caspase-8 by shRNA to prevent TEC apoptosis and IRI was evaluated. IRI was induced by renal artery clamping for 45 or 60 min at 32°C in uninephrectomized C57BL/6 mice. Here, we showed that Fas or pro-caspase-8 expression was significantly knocked down in TEC by stable expression of shRNA, resulting in resistance to apoptosis induced by superoxide, IFN-,/TNF-, and anti-Fas antibody. Inferior vena cava delivery of pHEX-small interfering RNA targeting Fas or pro-caspase-8 resulted in protection of kidney from IRI, indicated by reduction of renal tubular injury (necrosis and apoptosis) and serum creatinine or blood urea nitrogen. Our data suggest that shRNA-based therapy targeting Fas and caspase-8 in renal cells can lead to protection of kidney from IRI. Attenuation of pro-apoptotic proteins using genetic manipulation strategies such as shRNA might represent a novel strategy to promote kidney allograft survival from rejection or failure. [source] Comparison of bovine RNA polymerase III promoters for short hairpin RNA expressionANIMAL GENETICS, Issue 4 2006L. S. Lambeth Summary RNA interference (RNAi) mediated by DNA-based expression of short hairpin RNA (shRNA) is a powerful method of sequence-specific gene knockdown. A number of vectors for expression of shRNA have been developed that feature promoters from RNA polymerase III (pol III)-transcribed genes of mouse or human origin. To advance the use of RNAi as a tool for functional genomic research and for future development of specific therapeutics in the bovine species, we have developed shRNA expression vectors that feature novel bovine RNA pol III promoters. We characterized two bovine U6 small nuclear RNA (snRNA) promoters (bU6-2 and bU6-3) and a bovine 7SK snRNA promoter (b7SK). We compared the efficiency of each of these promoters to express shRNA molecules. Promoter activity was measured in the context of RNAi by targeting and suppressing the reporter gene encoding enhanced green fluorescent protein. Results show that the b7SK promoter induced the greatest level of suppression in a range of cell lines. The comparison of these bovine promoters in shRNA expression is an important component for the future development of bovine-specific RNAi-based research. [source] Matrix metalloproteinase 13 loss associated with impaired extracellular matrix remodeling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factorsARTHRITIS & RHEUMATISM, Issue 8 2010Rosa Maria Borzí Objective To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. Methods MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and ,-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. Results Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, ,-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13,remodeled or ,degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. Conclusion MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors. [source] Involvement of MAPKs and NF-,B in tumor necrosis factor ,,induced vascular cell adhesion molecule 1 expression in human rheumatoid arthritis synovial fibroblastsARTHRITIS & RHEUMATISM, Issue 1 2010Shue-Fen Luo Objective To investigate the roles of MAPKs and NF-,B in tumor necrosis factor , (TNF,),induced expression of vascular cell adhesion molecule 1 (VCAM-1) in human rheumatoid arthritis synovial fibroblasts (RASFs). Methods Human RASFs were isolated from synovial tissue obtained from patients with RA who underwent knee or hip surgery. The involvement of MAPKs and NF-,B in TNF,-induced VCAM-1 expression was investigated using pharmacologic inhibitors and transfection with short hairpin RNA (shRNA) and measured using Western blot, reverse transcriptase,polymerase chain reaction, and gene promoter assay. NF-,B translocation was determined by Western blot and immunofluorescence staining. The functional activity of VCAM-1 was evaluated by lymphocyte adhesion assay. Results TNF,-induced VCAM-1 expression, phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK, and translocation of NF-,B were attenuated by the inhibitors of MEK-1/2 (U0126), p38 (SB202190), JNK (SP600125), and NF-,B (helenalin) or by transfection with their respective shRNA. TNF,-stimulated translocation of NF-,B into the nucleus and NF-,B promoter activity were blocked by Bay11-7082, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNF, in RASFs transfected with VCAM-1-Luc, and this promoter activity was inhibited by Bay11-7082, U0126, SB202190, and SP600125. Moreover, up-regulation of VCAM-1 increased the adhesion of lymphocytes to the RASF monolayer, and this adhesion was attenuated by pretreatment with helenalin, U0126, SP600125, or SB202190 prior to exposure to TNF, or by anti,VCAM-1 antibody before the addition of lymphocytes. Conclusion In RASFs, TNF,-induced VCAM-1 expression is mediated through activation of the p42/p44 MAPK, p38 MAPK, JNK, and NF-,B pathways. These results provide new insights into the mechanisms underlying cytokine-initiated joint inflammation in RA and may inspire new targeted therapeutic approaches. [source] Transmembrane BAFF from rheumatoid synoviocytes requires interleukin-6 to induce the expression of recombination-activating gene in B lymphocytesARTHRITIS & RHEUMATISM, Issue 5 2009Caroline Rochas Objective B cells that accumulate in the synovial tissue of rheumatoid arthritis (RA) patients revise their receptors due to coordinate expression of recombination-activating gene 1 (RAG-1) and RAG-2 genes. The aim of this study was to determine the mechanisms that control this re-expression. Methods B cells from healthy control subjects were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA and osteoarthritis (OA). Re-expression of RAG messenger RNA (mRNA) and proteins was analyzed by reverse transcription,polymerase chain reaction (RT-PCR) and indirect immunofluorescence. Activity of RAG enzymes was evaluated by flow cytometry to measure variations in immunoglobulin , and , light chain expression and by ligation-mediated,PCR to assess specific DNA breaks. Blocking antibodies, short hairpin RNA, and recombinant cytokine were used to identify the molecules involved in RAG re-expression. Results RA FLS, but not OA FLS, induced B cells to re-express RAG mRNA and proteins. Enzymes were functional, since the ,-to-, ratios decreased and specific DNA breaks were detectable after coculture with RA FLS. Transmembrane BAFF provided the first signal of RAG re-expression, since its down-regulation in RA FLS prevented RAG gene transcription in B cells. The failure of transmembrane BAFF from OA FLS to induce RAG suggests that a second signal was provided by RA FLS. Interleukin-6 (IL-6) is a candidate, since blockade of its receptors precluded transcription of RAG genes by RA FLS. Unless supplemented with IL-6, OA FLS were unable to induce RAG gene expression in normal B cells. Conclusion Two independent signals are required for the induction of RAG gene expression in B cells that infiltrate the synovium of patients with RA. [source] The ,7 nicotinic acetylcholine receptor on fibroblast-like synoviocytes and in synovial tissue from rheumatoid arthritis patients: A possible role for a key neurotransmitter in synovial inflammationARTHRITIS & RHEUMATISM, Issue 5 2009Marjolein A. Van Maanen Objective Recent studies have suggested an important role for neurotransmitters as modulators of inflammation. Therefore, we undertook this study to investigate the expression of the ,7 subunit of the nicotinic acetylcholine receptor (,7nAChR) and its function in rheumatoid arthritis (RA). Methods The potential role of the ,7nAChR in modulating proinflammatory cytokine expression in fibroblast-like synoviocytes (FLS) was identified by screening an adenoviral short hairpin RNA (Ad.shRNA) library. An ,7-specific antibody was used for immunohistochemistry, and fluorescein isothiocyanate,labeled ,-bungarotoxin, which binds specifically to the ,7nAChR, was used for immunofluorescence. Gene expression in FLS was determined by quantitative polymerase chain reaction with primers specific for the ,7nAChR. In addition, we analyzed messenger RNA (mRNA) expression of dup,7, a variant ,7 transcript. Next, we studied the functional role of the ,7nAChR in RA FLS by examining the effects of ,7-specific agonists on the production of interleukin-6 (IL-6) and IL-8 by activated FLS. Results A screen using an Ad.shRNA library against 807 transcripts revealed that a specific ,7nAChR shRNA potently modulated IL-8 and matrix metalloproteinase expression in FLS. The ,7nAChR was expressed in the inflamed synovium from RA patients, predominantly in the intimal lining layer. We found ,7nAChR expression at both the mRNA and protein level in cultured RA FLS. FLS also constitutively expressed dup,7 mRNA. Specific ,7nAChR agonists reduced tumor necrosis factor ,,induced IL-6 and IL-8 production by FLS. Conclusion The ,7nAChR and its dup,7 variant are expressed in RA synovium, where they may play a critical role in regulating inflammation. Targeting the ,7nAChR could provide a novel antiinflammatory approach to the treatment of RA. [source] Involvement of protein kinase C, in interleukin-1, induction of ADAMTS-4 and type 2 nitric oxide synthase via NF-,B signaling in primary human osteoarthritic chondrocytesARTHRITIS & RHEUMATISM, Issue 12 2007Priya S. Chockalingam Objective Protein kinase C, (PKC,), an atypical PKC, has been found to be transcriptionally up-regulated in human osteoarthritic (OA) articular cartilage. This study was undertaken to examine the role of PKC, in interleukin-1, (IL-1,),induced NF-,B signaling in human OA chondrocytes, and ultimately to better understand its function in the regulation of downstream mediators of cartilage matrix degradation. Methods Pharmacologic inhibitors or genetic knockdown techniques were used to investigate the role of PKC,. Western blot analysis was used to evaluate phosphorylation of PKC, and NF-,B. Quantitative polymerase chain reaction (PCR) and activity assays were used to evaluate ADAMTS-4 expression and aggrecanase activity, respectively. Quantitative PCR, biochemical identification, and Western blot analysis were used to evaluate type 2 nitric oxide synthase (NOS2) and NO production. Results Phosphorylation of PKC, and NF-,B was induced by IL-1, treatment in a time-dependent manner, and was specifically inhibited by inhibitors of atypical PKCs. Inhibition of PKC, suppressed IL-1,,induced up-regulation of ADAMTS-4 messenger RNA (mRNA) and aggrecanase activity. Inhibitors of atypical PKCs also inhibited IL-1,,induced NO production and NOS2 mRNA expression, demonstrating a novel link between PKC, and NO production. Furthermore, small interfering RNA, or short hairpin RNA,mediated knockdown of PKC, mRNA resulted in significant repression of both ADAMTS-4 and NOS2 mRNA expression. Conclusion Our results show that PKC, is involved in the regulation of IL-1,,induced NF-,B signaling in human OA chondrocytes, which in turn regulates downstream expression of ADAMTS-4 and NOS2. Therefore, inhibition of PKC, could potentially regulate the production of matrix-degrading enzymes as well as NO production and have a profound effect on disease progression in OA. [source] Therapeutic potential of RNA interference against cancerCANCER SCIENCE, Issue 8 2006Fumitaka Takeshita One of the most dramatic events of the past 5 years in the field of molecular biology has been the discovery of RNA interference (RNAi). Although RNAi is an evolutionarily conserved phenomenon for sequence-specific gene silencing in mammalian cells, exogenous small interfering RNA (siRNA) and vector-based short hairpin RNA (shRNA) can also invoke RNAi responses. Both are now not only experimental tools for analyzing gene function but are expected to be excellent avenues for drug target discovery and the emerging class of gene medicine for targeting incurable diseases such as cancer. The success of cancer therapeutic use of RNAi relies on the development of safe and efficacious delivery systems that introduce siRNA and shRNA expression vectors into target tumor cells. For their delivery, a variety of strategies have been used, most of them based on traditional gene therapy delivery systems. In this review, we present siRNA delivery method strategies and discuss the potential of RNAi-based gene therapy in cancer treatment. (Cancer Sci 2006; 97: 689,696) [source] Caveolin-1 influences P2X7 receptor expression and localization in mouse lung alveolar epithelial cellsFEBS JOURNAL, Issue 12 2007K. Barth The P2X7 receptor has recently been described as a marker for lung alveolar epithelial type I cells. Here, we demonstrate both the expression of P2X7 protein and its partition into lipid rafts in the mouse lung alveolar epithelial cell line E10. A significant degree of colocalization was observed between P2X7 and the raft marker protein Caveolin-1; also, P2X7 protein was associated with caveolae. A marked reduction in P2X7 immunoreactivity was observed in lung sections prepared from Caveolin-1-knockout mice, indicating that Caveolin-1 expression was required for full expression of P2X7 protein. Indeed, suppression of Caveolin-1 protein expression in E10 cells using short hairpin RNAs resulted in a large reduction in P2X7 protein expression. Our data demonstrate a potential interaction between P2X7 protein and Caveolin-1 in lipid rafts, and provide a basis for further functional and biochemical studies to probe the physiologic significance of this interaction. [source] Stringent testing identifies highly potent and escape-proof anti-HIV short hairpin RNAsTHE JOURNAL OF GENE MEDICINE, Issue 6 2009Karin J. von Eije Abstract Background RNA interference (RNAi) is a cellular mechanism that can be induced by small interfering RNAs to mediate sequence-specific gene silencing by cleavage of the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short hairpin RNAs (shRNAs). Previously, we used a co-transfection assay in which shRNA constructs were transfected with an HIV-1 molecular clone to identify 20 shRNA inhibitors that target highly conserved HIV-1 sequences. Methods In the present study, we selected the most potent shRNAs to formulate a combinatorial shRNA therapy and determine the best and easiest method for antiviral shRNA selection. We performed transient inhibition assays with either a luciferase reporter or HIV-1 molecular clone and also infected shRNA-expressing T cell lines with HIV-1 and monitored virus replication. The latter assay allows detection of viral escape. In addition, we also tested shRNA-expressing T cells upon challenge with increasing dosages of HIV-1, and measured the dose required to result in massive virus-induced syncytia formation in this 2-week assay. Results Extended culturing selected three highly effective shRNAs that do not allow viral replication for more than 100 days. This difference in potency was not observed in the transient co-transfection assays. The use of increased dosages of HIV-1 selected the same highly potent shRNAs as the laborious and extended escape study. Conclusions These highly potent shRNAs could be used for a clinical vector and the comparison of the developed assays might help other researchers in their search for antiviral shRNAs. Copyright © 2009 John Wiley & Sons, Ltd. [source] A retrovirus-based system to stably silence GDF-8 expression and enhance myogenic differentiation in human rhabdomyosarcoma cellsTHE JOURNAL OF GENE MEDICINE, Issue 8 2008Zhuo Yang Abstract Background Myostatin, also called GDF-8, a secreted growth and differentiating factor that belongs to the transforming growth factor-, superfamily, is a known negative regulator of myogenesis in vivo. Overexpression of GDF-8 contributes to the lack of differentiation in human rhabdomyosarcoma (RMS) cells. We investigated whether a retrovirus-based RNA interference (RNAi) system against GDF-8 expression in human RMS cells would enhance myogenic differentiation. Methods A retrovirus-based RNAi system was developed that utilized the U6-RNA polymerase III promoter to drive efficient expression and deliver the GDF8-specific short hairpin RNAs (shRNAs) in human RMS cell A204. In this system, the retrovirus vector was integrated into the host cell genome and allowed stable expression of shRNAs. GDF-8 expression was determined by real-time polymerase chain reaction and western blotting analysis. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the cell proliferation. Myogenic differentiation markers were monitored by western blotting analysis. Cell cycle and apoptosis was determined by propidium iodide staining and analysed in a flow cytometer. Results In the siGDF8 A204 cell pools, the levels of both GDF-8 mRNA and protein were dramatically reduced by this RNAi system. In differentiation conditions, inhibition of myostatin synthesis led to enhanced cell cycle withdrawal, consequently stimulated myogenic differentiation and increased the rate of tumor cell apoptosis. Conclusions The results demonstrate that deactivation of myostatin by using retrovirus-based RNAi thus may be useful for therapy in rhabdomyosarcomas. Copyright © 2008 John Wiley & Sons, Ltd. [source] Oncostatin M,induced CCL2 transcription in osteoblastic cells is mediated by multiple levels of STAT-1 and STAT-3 signaling: An implication for the pathogenesis of arthritisARTHRITIS & RHEUMATISM, Issue 5 2009Sang-Heng Kok Objective To examine the roles of STATs 1 and 3 in CCL2 production in human osteoblastic cells and their influences on arthritis development. Methods The expression of CCL2 in primary human osteoblasts and U2OS human osteoblastic cells was examined by Northern blotting and enzyme-linked immunosorbent assay. The roles of STAT-1/3 and c-Fos were assessed using short hairpin RNAs (shRNA) to silence their functions. Serine phosphorylation of STATs was assessed by Western blotting. Promoter activities of c-Fos and CCL2 were assessed by chloramphenicol acetyltransferase and luciferase assays, respectively. Interactions of STAT-1, STAT-3, and c-Fos with DNA were evaluated by electrophoretic mobility shift assay (EMSA) and immunoprecipitation. The effect of the JAK inhibitor AG-490 on collagen-induced arthritis (CIA) in rats was examined using immunohistochemistry. Results Oncostatin M (OSM) stimulated CCL2 expression in primary human osteoblasts and U2OS cells. In U2OS cells, STAT-1 and STAT-3 were involved in OSM-stimulated CCL2 expression, and both the phosphatidylinositol 3-kinase/Akt and MEK/ERK pathways were implicated in the activation of these STATs. STAT-1 and STAT-3 modulated the expression of c-Fos and directly transactivated the CCL2 promoter. Moreover, EMSA showed formation of a DNA,protein complex containing STAT-1, STAT-3, and interestingly, c-Fos. Immunoprecipitation confirmed the binding between c-Fos and STAT-1/3. Reporter assay revealed synergistic attenuation of CCL2 promoter activity by shRNA targeting of STAT-1, STAT-3, and c-Fos. AG-490 suppressed OSM-stimulated activation of STAT-1/3 and synthesis of CCL2 in vitro and diminished the severity of CIA and the number of CCL2-synthesizing osteoblasts in vivo. Conclusion These findings show that multiple levels of STAT-1/3 signaling modulate OSM-stimulated CCL2 expression in human osteoblastic cells. Clinically, this pathway may be related to the pathogenesis of arthritis. [source] | ||||||||||||||||