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Short Interfering RNAs (short + interfering_rna)
Selected AbstractsInterferon alpha receptors are important for antiproliferative effect of interferon-, against human hepatocellular carcinoma cellsHEPATOLOGY RESEARCH, Issue 1 2007Bazarragchaa Damdinsuren Aim:, Interferon (IFN)-, is a promising drug for the prevention and treatment of hepatocellular carcinoma (HCC). We reported that responders to IFN-,/5-fluorouracil combination therapy expressed higher IFN alpha receptor (IFNAR)2 in tumor. Herein we studied involvement of IFNARs in response to IFN-, in HCC cells. Methods:, IFN-, sensitivity and expression of IFNARs were studied in six HCC cell lines (HuH7, PLC/PRF/5, HLE, HLF, HepG2, Hep3B) using growth-inhibitory and RT-PCR, Western blot assays. Short interfering RNAs (SiRNAs) against IFNAR1 and 2 were used to analyze the role of the IFNARs in IFN-,'s effect and signal transduction. Results:, The expressions of IFNAR1 and 2c mRNAs were higher in PLC/PRF/5 cells than those in other cell lines, and PLC/PRF/5 cells expressed abundant IFNAR2c on their cell membrane. When we examined the sensitivity of the HCC cell lines to the growth-inhibitory effect of IFN-,, PLC/PRF/5 exhibited a significant response, while the other cells were much more resistant. Knockdown of either IFNAR1 or 2 using siRNAs suppressed the IFN-,'s signal transduction (2.5-fold), and decreased the growth-inhibitory effect (down by 69.9% and 67.3%). Conclusion:, The results suggest that the expression of IFNAR1 and IFNAR2c independently are important for the antiproliferative effect of IFN-, in HCC cells. [source] Novel siRNA-based molecular beacons for dual imaging and therapyBIOTECHNOLOGY JOURNAL, Issue 4 2007Emmanuel Chang Short interfering RNAs (siRNAs) have become a mainstream tool reliably used to study and silence protein expression. We offer a proof-of-principle demonstration that siRNAs may be modified into a siRNA-based molecular beacon that activates upon binding to sequence-specific mRNA in cells while mediating RNA interference. We successfully demonstrate detection and knockdown of telomerase expression in human breast cancer cells. This probe provides a novel look at siRNA target validation that is not currently possible in live cells and holds promising potential in biological applications for disease detection and therapy based on mRNA expression, such as a telomerase-targeted siRNA probe in cancer. [source] Down-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell linesFEBS JOURNAL, Issue 23 2006Yuanying Ding Intracellular heme concentrations are maintained in part by heme degradation, which is catalyzed by heme oxygenase. Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2. Recent studies have identified HO-2 as a potential oxygen sensor. To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expression profiles of HO-2 and the biochemical consequences of HO-2 knockdown with specific short interfering RNA (siRNA) in human cells. Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma. Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells. In contrast, knockdown of HO-1 expression did not noticeably influence HO-2 expression. HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells. Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner. Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin. HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis. These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2. [source] Phosphoinositide 3-kinase signalling in lung disease: leucocytes and beyondIMMUNOLOGY, Issue 4 2007David A. Medina-Tato Summary The family of lipid kinases termed phosphoinositide-3-kinase (PI3K) is known to contribute at multiple levels to innate and adaptive immune responses, and is hence an attractive target for drug discovery in inflammatory and autoimmune disease, including respiratory diseases. The development of isoform-selective pharmacological inhibitors, targeted gene manipulation and short interfering RNA (siRNA) target validation have facilitated a better understanding of the role that each member of this family of kinases plays in the physiology and pathology of the respiratory system. In this review, we will evaluate the evidence for the roles of specific PI3K isoforms in the lung and airways, and discuss their potential as targets for novel drug therapies. [source] Sp1 and Smad3 are required for high glucose-induced p21WAF1 gene transcription in LLC-PK1 cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2007Tsai-Der Chuang Abstract The cyclin-dependent kinase inhibitor p21WAF1 is required for diabetic glomerular hypertrophy. High glucose-induced hypertrophy in proximal tubule cells is dependent on transforming growth factor-, (TGF-,). Many of the TGF-,-induced effects are dependent on Smad2/3. Thus, the molecular mechanisms of high glucose-induced p21WAF1 and hypertrophy were studied in high glucose-cultured proximal tubule-like LLC-PK1 cells. We found that high glucose (30 mM) induced hypertrophy at 72 h. High glucose also increased the expression of p21WAF1 protein and p21WAF1 mRNA transcription and abundance at 48 h. The DNA element in the 5, regulatory region of p21WAF1 gene essential for high glucose-induced p21WAF1 gene transcription was identified as Sp1 by a series of the 5, regulatory region of p21WAF1 gene deletion mutants. Moreover, high glucose activated Smad2/3 while increasing the Sp1 DNA-binding activity. High glucose also increased the Sp1-dependent transcriptional activity of p21WAF1 gene. High glucose-induced hypertrophy was attenuated by p21WAF1 short interfering RNA and Smad3 dominant-negative plasmid transfection. We concluded that high glucose induced hypertrophy via Sp1-Smad2/3-dependent activation of p21WAF1 gene transcription in LLC-PK1 cells. J. Cell. Biochem. 102: 1190,1201, 2007. © 2007 Wiley-Liss, Inc. [source] Cancer, chitosan nanoparticles and catalytic nucleic acidsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 1 2009Mei Lin Tan Abstract Objectives The aim of this review was to examine gene therapy involving DNAzyme and siRNA encapsulation into chitosan nanoparticles, discussing the current and future status of this drug delivery system in enhancing drug delivery and cancer therapy. Key findings Cancer is a disease state in which the cells in our body undergo mutations at the genetic level and are transformed, acquiring the ability to replicate limitlessly. Conventional cancer treatment involves the use of surgery and cytotoxic chemotherapy and/or radiotherapy, which have the potential of harming normal, otherwise healthy, non-neoplastic cells. Newer forms of therapy such as immunotherapy and gene therapy have shown initial promise, but still require better ways to limit exposure to cancerous lesions in the body. As a result drug delivery systems have been developed in attempts to deliver therapeutics specifically to the target lesion site. One recent drug delivery system has revolved around the use of chitosan nanoparticle technology, where therapeutics are encapsulated into nanoparticles and targeted to tumours. Summary Though few, attempts at encapsulating therapeutics such as deoxyribozymes and small or short interfering RNA have been optimistic and encouraging. [source] Efficient suppression of murine arthritis by combined anticytokine small interfering RNA lipoplexesARTHRITIS & RHEUMATISM, Issue 8 2008Maroun Khoury Objective Blocking tumor necrosis factor (TNF) effectively inhibits inflammation and joint damage in rheumatoid arthritis (RA), but 40% of RA patients respond only transiently or not at all to the current anti-TNF biotherapies. The purpose of this study was to develop an alternative targeted therapy for this subgroup of RA patients. As proof of concept, we tested the efficiency of an RNA interference (RNAi),based intervention that targets proinflammatory cytokines in suppressing murine collagen-induced arthritis (CIA). Methods Two synthetic short interfering RNA (siRNA) sequences were designed for each of the proinflammatory cytokines interleukin-1 (IL-1), IL-6, and IL-18. Their silencing specificity was assessed according to lipopolysaccharide-induced messenger RNA expression in J774.1 mouse macrophages as compared with control siRNA. For in vivo administration, siRNA were formulated as lipoplexes with the RPR209120/DOPE liposome and a carrier DNA and were injected intravenously (0.5 mg/kg) into DBA/1 mice with CIA. Results Weekly injections of anti,IL-1, anti,IL-6, or anti,IL-18 siRNA-based lipoplexes significantly reduced the incidence and severity of arthritis, abrogating joint swelling and destruction of cartilage and bone, both in the preventative and the curative settings. The most striking therapeutic effect was observed when the 3 siRNA were delivered in combination. The siRNA lipoplex cocktail reduced all pathologic features of RA, including inflammation, joint destruction, and the Th1 response, and overall parameters of RA were improved as compared with anti-TNF siRNA lipoplex,based treatment. Conclusion Our results present a novel option for in vivo RNAi-based antiinflammatory immunotherapy. Our findings indicate that intravenous administration of a lipoplex cocktail containing several anticytokine siRNA is a promising novel antiinflammatory therapy for RA, as well as a useful and simple tool for understanding the pathophysiology of RA and for evaluating new therapeutic candidates. [source] Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81,HEPATOLOGY, Issue 6 2007Mirjam B. Zeisel Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR-BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR-BI for productive HCV infection remains unclear. In this study, we investigated the role of SR-BI as an entry factor for infection of human hepatoma cells using cell culture,derived HCV (HCVcc). Anti,SR-BI antibodies directed against epitopes of the human SR-BI extracellular loop specifically inhibited HCVcc infection in a dose-dependent manner. Down-regulation of SR-BI expression by SR-BI,specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR-BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81,HCV interaction. Although the addition of high-density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti,SR-BI antibodies and SR-BI,specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR-BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV,CD81 interaction. (HEPATOLOGY 2007.) [source] Pml and TAp73 interacting at nuclear body mediate imatinib-induced p53-independent apoptosis of chronic myeloid leukemia cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2009Jin-Hwang Liu Abstract Bcr-abl signals for leukemogenesis of chronic myeloid leukemia (CML) and activates ras. Since the function of promyelocytic leukemia protein (pml) is provoked by ras to promote apoptosis and senescence in untransformed cells, the function is probably masked in CML. Imatinib specifically inhibits bcr-abl and induces apoptosis of CML cells. As reported previously, p53wild CML was more resistant to imatinib than that lacking p53. Here, we searched for an imatinib-induced p53 independent proapoptotic mechanism. We found imatinib up-regulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), checkpoint kinase 2 (chk2) and transactivation-competent (TA) p73; expression of pml and bax; formation of PML-nuclear body (NB); and co-localization of TAp73/PML-NB in p53-nonfunctioning K562 and p53mutant Meg-01 CML cells, but not in BCR-ABL - HL60 cells. In K562 cells, with short interfering RNAs (siRNAs), knockdown of pml led to dephosphorylation of TAp73. Knockdown of either pml or TAp73 abolished the imatinib-induced apoptosis. Inhibition of p38 MAPK with SB203580 led to dephosphorylation of TAp73, abolishment of TAp73/PML-NB co-localization, and the subsequent apoptosis. Conversely, interferon ,-2a (IFN,), which increased phosphrylated TAp73 and TAp73/PML-NB co-localization, increased additively apoptosis with imatinib. The imatinib-induced TAp73/PML-NB co-localization was accompanied by co-immpunoprecipitation of TAp73 with pml. The imatinib-induced co-localization was also found in primary CML cells from 3 of 6 patients, including 2 with p53mutant and one with p53wild. A novel p53-independent proapoptotic mechanism using p38 MAPK /pml/TAp73 axis with a step processing at PML-NB and probably with chk2 and bax being involved is hereby evident in some imatinib-treated CML cells. © 2009 UICC [source] RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA constructMOLECULAR PLANT PATHOLOGY, Issue 4 2009NORA SCHWIND SUMMARY Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (,)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses. [source] Viroid-induced RNA silencing of GFP-viroid fusion transgenes does not induce extensive spreading of methylation or transitive silencingTHE PLANT JOURNAL, Issue 1 2004Ulrike Vogt Summary Viroid infection is associated with the production of short interfering RNAs (siRNAs), a hallmark of post-transcriptional gene silencing (PTGS). However, viroid RNAs autonomously replicating in the nucleus have not been shown to trigger the degradation of homologous RNA in the cytoplasm. To investigate the potential of viroids for the induction of gene silencing, non-infectious fragments of potato spindle tuber viroid (PSTVd) cDNA were transcriptionally fused to the 3, end of the green fluorescent protein (GFP)-coding region. Introduction of such constructs into tobacco plants resulted in stable transgene expression. Upon PSTVd infection, transgene expression was suppressed and partial de novo methylation of the transgene was observed. PSTVd-specific siRNA was detected but none was found corresponding to the gfp gene. Methylation was restricted almost entirely to the PSTVd-specific part of the transgene. Neither a gfp transgene construct lacking viroid-specific elements was silenced nor was de novo methylation detected, when it was introduced into the genetic background of the PSTVd-infected plant lines containing silenced GFP:PSTVd transgenes. The absence of gfp -specific siRNAs and of significant methylation within the gfp -coding region demonstrated that neither silencing nor DNA methylation spread from the initiator region into adjacent 5, regions. [source] Crystallization and preliminary X-ray analysis of the C-terminal RNase III domain of human DicerACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2006Daijiro Takeshita Human Dicer protein contains two RNase III domains (RNase IIIa and RNase IIIb) which are involved in the production of short interfering RNAs (siRNAs). The C-terminal RNase III domain (RNase IIIb) of human Dicer was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. The crystals belonged to space group C2221, with unit-cell parameters a = 88.6, b = 199.7, c = 119.6,Å, and diffracted X-rays to 2.0,Å resolution. The asymmetric unit contained three molecules of the RNase IIIb and the solvent content was 67%. [source] Induction of insulin-like growth factor-I by interleukin-17F in bronchial epithelial cellsCLINICAL & EXPERIMENTAL ALLERGY, Issue 7 2010M. Kawaguchi Summary Cite this as: M. Kawaguchi, J. Fujita, F. Kokubu, G. Ohara, S-K Huang, S. Matsukura, Y. Ishii, M. Adachi, H. Satoh and N. Hizawa, Clinical & Experimental Allergy, 2010 (40) 1036,1043. Background Increased expression of IL-17F has been noted in the airway of asthmatic patients, but its role in asthma has not been fully elucidated. Insulin-like growth factor-I (IGF-I) is known to be involved in airway remodelling and inflammation, while its regulatory mechanisms remain to be defined. Objective To further clarify the biological function of IL-17F, we investigated whether IL-17F is able to regulate the expression of IGF-I in bronchial epithelial cells. Methods Bronchial epithelial cells were stimulated with IL-17F in the presence or absence of T-helper type 2 cytokines. Various kinase inhibitors were added to the culture to identify the key signalling events leading to the expression of IGF-I, in conjunction with the use of short interfering RNAs (siRNAs) targeting mitogen- and stress-activated protein kinase (MSK) 1, p90 ribosomal S6 kinase (p90RSK), and cyclic AMP response element-binding protein (CREB). Results IL-17F significantly induced IGF-I gene and protein expression, and co-stimulation with IL-4 and IL-13 augmented its production. MAP kinase kinase (MEK) inhibitors and the Raf1 kinase inhibitor significantly inhibited IGF-I production, and the combination of PD98059 and Raf1 kinase inhibitor showed further inhibition. Overexpression of Raf1 and Ras dominant-negative mutants inhibited its expression. MSK1 inhibitors significantly blocked IL17F-induced IGF-I expression. Moreover, transfection of the siRNAs targeting MSK1, p90RSK, and CREB blocked its expression. Conclusions In bronchial epithelial cells, IL-17F is able to induce the expression of IGF-I via the Raf1,MEK1/2,ERK1/2,MSK1/p90RSK,CREB pathway in vitro. [source] | ||||||||||||||||