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Small Interfering RNA (small + interfering_rna)
Kinds of Small Interfering RNA Selected AbstractsGlucagon-like peptide-1 receptor is present on human hepatocytes and has a direct role in decreasing hepatic steatosis in vitro by modulating elements of the insulin signaling pathway,HEPATOLOGY, Issue 5 2010Nitika Arora Gupta Glucagon-like peptide 1 (GLP-1) is a naturally occurring peptide secreted by the L cells of the small intestine. GLP-1 functions as an incretin and stimulates glucose-mediated insulin production by pancreatic , cells. In this study, we demonstrate that exendin-4/GLP-1 has a cognate receptor on human hepatocytes and that exendin-4 has a direct effect on the reduction of hepatic steatosis in the absence of insulin. Both glucagon-like peptide 1 receptor (GLP/R) messenger RNA and protein were detected on primary human hepatocytes, and receptor was internalized in the presence of GLP-1. Exendin-4 increased the phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK-1), AKT, and protein kinase C , (PKC-,) in HepG2 and Huh7 cells. Small interfering RNA against GLP-1R abolished the effects on PDK-1 and PKC-,. Treatment with exendin-4 quantitatively reduced triglyceride stores compared with control-treated cells. Conclusion: This is the first report that the G protein,coupled receptor GLP-1R is present on human hepatocytes. Furthermore, it appears that exendin-4 has the same beneficial effects in vitro as those seen in our previously published in vivo study in ob/ob mice, directly reducing hepatocyte steatosis. Future use for human nonalcoholic fatty liver disease, either in combination with dietary manipulation or other pharmacotherapy, may be a significant advance in treatment of this common form of liver disease. (HEPATOLOGY 2010) [source] HuR regulates gap junctional intercellular communication by controlling ,-catenin levels and adherens junction integrity,HEPATOLOGY, Issue 5 2009Niloofar Ale-Agha Gap junctional intercellular communication (GJIC) plays a critical role in the regulation of tissue homeostasis and carcinogenesis and is modulated by the levels, subcellular localization, and posttranslational modification of gap junction proteins, the connexins (Cx). Here, using oval cell-like rat liver epithelial cells, we demonstrate that the RNA-binding protein HuR promotes GJIC through two mechanisms. First, HuR silencing lowered the levels of Cx43 protein and Cx43 messenger RNA (mRNA), and decreased Cx43 mRNA half-life. This regulation was likely due to the direct stabilization of Cx43 mRNA by HuR, because HuR associated directly with Cx43 mRNA, a transcript that bears signature adenylate-uridylate-rich (AU-rich) and uridylate-rich (U-rich) sequences in its 3,-untranslated region. Second, HuR silencing reduced both half-life and the levels of ,-catenin mRNA, also a target of HuR; accordingly, HuR silencing lowered the levels of whole-cell and membrane-associated ,-catenin. Coimmunoprecipitation experiments showed a direct interaction between ,-catenin and Cx43. Small interfering RNA (siRNA)-mediated depletion of ,-catenin recapitulated the effects of decreasing HuR levels: it attenuated GJIC, decreased Cx43 levels, and redistributed Cx43 to the cytoplasm, suggesting that depletion of ,-catenin in HuR-silenced cells contributed to lowering Cx43 levels at the membrane. Finally, HuR was demonstrated to support GJIC after exposure to a genotoxic agent, doxorubicin, or an inducer of differentiation processes, retinoic acid, thus pointing to a crucial role of HuR in the cellular response to stress and in physiological processes modulated by GJIC. Conclusion: HuR promotes gap junctional intercellular communication in rat liver epithelial cells through two related regulatory processes, by enhancing the expression of Cx43 and by increasing the expression of ,-catenin, which, in turn, interacts with Cx43 and is required for proper positioning of Cx43 at the plasma membrane. (HEPATOLOGY 2009.) [source] MicroRNA in the immune system, microRNA as an immune systemIMMUNOLOGY, Issue 3 2009Li-Fan Lu Summary The advent of microRNA has potentially uncovered a new level of complexity to be considered for every biological process. Through the modulation of transcription and translation, microRNA alter the basal state of cells and the outcome of stimulatory events. The exact effect of the microRNA network and individual microRNA on cellular processes is only just starting to be dissected. In the immune system, microRNA appear to have a key role in the early differentiation and effector differentiation of B cells. In T cells, microRNA have been shown to be key regulators of the lineage induction pathways, and to have a strong role in the induction, function and maintenance of the regulatory T-cell lineage. MicroRNA are also important for regulating the differentiation of dendritic cells and macrophages via toll-like receptors, with responsibilities in suppressing effector function before activation and enhancing function after stimulation. In addition to regulating key processes in the immune system, microRNA may also represent an archaic immune system themselves. Small interfering RNA of viral origin has been shown to function as an intracellular mediator in the suppression of viral infection in eukaryotes as diverse as plants, insects, nematodes and fungi, and there is growing evidence that endogenous mammalian microRNA can have similar impacts. In this article we speculate that the anti-viral function of microRNA drove the expression of different subsets of microRNA in different cellular lineages, which may have, in turn, led to the myriad of roles microRNA play in lineage differentiation and stability. [source] Small interfering RNA (siRNA) inhibits the expression of the Her2/neu gene, upregulates HLA class I and induces apoptosis of Her2/neu positive tumor cell linesINTERNATIONAL JOURNAL OF CANCER, Issue 1 2004Aniruddha Choudhury Abstract Silencing of a specific mRNA using double stranded RNA oligonucleotides represents one of the newest technologies for suppressing a specific gene product. Small interfering RNA (siRNA) are 21 nucleotides long, double stranded RNA fragments that are identical in sequence to the target mRNA. We designed 3 such siRNA against the Her2/neu (HER2) gene. The HER2 gene is known to play an important role in the oncogenesis of several types of cancers, such as breast, ovarian, colon and gastric cancers. Introduction of the siRNA into HER2 positive tumor lines in vitro greatly reduced the cell surface expression of the HER2 protein. Concurrently, a range of effects on cell physiology, such as growth inhibition or apoptosis, was observed. The expression of HLA class I was observed to be upregulated when HER2 was silenced with siRNA. Treatment of SKBr3 and MCF7/HER2 tumor cell lines with the HER2 siRNA resulted in growth arrest of cells in the late G1/S-phase. Our results suggest that siRNA may be an effective method of abrogating the effect of HER2 in tumorigenesis. © 2003 Wiley-Liss, Inc. [source] Cyclooxygenase-2 Expression and Prostaglandin E2 Production in Response to Acidic pH Through OGR1 in a Human Osteoblastic Cell Line,,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2008Hideaki Tomura Abstract Acidosis has been shown to induce depletion of bone calcium from the body. This calcium release process is thought to be partially cell mediated. In an organ culture of bone, acidic pH has been shown to induce cyclooxygenase-2 (COX-2) induction and prostaglandin E2 (PGE2) production, resulting in stimulation of bone calcium release. However, the molecular mechanisms whereby osteoblasts sense acidic circumstances and thereby induce COX-2 induction and PGE2 production remain unknown. In this study, we used a human osteoblastic cell line (NHOst) to characterize cellular activities, including inositol phosphate production, intracellular Ca2+ concentration ([Ca2+]i), PGE2 production, and COX-2 mRNA and protein expression, in response to extracellular acidification. Small interfering RNA (siRNA) specific to the OGR1 receptor and specific inhibitors for intracellular signaling pathways were used to characterize acidification-induced cellular activities. We found that extracellular acidic pH induced a transient increase in [Ca2+]i and inositol phosphate production in the cells. Acidification also induced COX-2 induction, resulting in PGE2 production. These proton-induced actions were markedly inhibited by siRNA targeted for the OGR1 receptor and the inhibitors for Gq/11 protein, phospholipase C, and protein kinase C. We conclude that the OGR1/Gq/11/phospholipase C/protein kinase C pathway regulates osteoblastic COX-2 induction and subsequent PGE2 production in response to acidic circumstances. [source] Inhibition of allergic responses by CD40 gene silencingALLERGY, Issue 3 2009M. Suzuki Background:, Gene silencing using small interfering RNA (siRNA) is a potent method of specifically knocking down molecular targets. Small interfering RNA is therapeutically promising, however, treatment of allergic diseases with siRNA has not been explored in vivo. The aim of this study was to evaluate therapeutic effects of CD40 siRNA on inhibition of allergic responses. Methods:, Mice sensitized with ovalbumin (OVA) and alum were treated with CD40 siRNA, scrambled siRNA, or phosphate buffer saline (PBS) alone, and then challenged intranasally with OVA. Results:, A significant reduction in nasal allergic symptoms was observed in the CD40 siRNA treated OVA-allergic mice compared to the controls of scrambled siRNA and PBS alone, which is correlated with the decrease of local eosinophil accumulation. CD40 siRNA treatment knocked down CD40 expression on dendritic cells (DCs) in vivo and impaired their antigen presenting function. Treatment with CD40 siRNA resulted in inhibition of OVA-specific T cell response and decrease of interleukin-4 (IL-4), IL-5, and interferon-, production from T cells stimulated with OVA. Administration of CD40 siRNA also suppressed CD40 expression on B cells, resulting in down-regulation of OVA-specific immunoglobulin E (IgE), IgG1, and IgG2a levels. Additionally, increased regulatory T cells were observed in the CD40 siRNA treated mice. Conclusions:, The present study demonstrates a novel therapeutic use for siRNA in allergy. CD40 siRNA attenuated allergy through inhibition of DC and B cell functions and generation of regulatory T (Treg) cells. [source] Local matrix metalloproteinase 2 gene knockdown in balloon-injured rabbit carotid arteries using nonviral-small interfering RNA transfectionTHE JOURNAL OF GENE MEDICINE, Issue 1 2009Hanna Hlawaty Abstract Background Small interfering RNA (siRNA) delivery is a promising approach for the treatment of cardiovascular diseases. Matrix metalloproteinase (MMP) 2 over-expression in the arterial wall has been implicated in restenosis after percutaneous coronary intervention, as well as in spontaneous atherosclerotic plaque rupture. We hypothesized that in vivo local delivery of siRNA targeted at MMP2 (MMP2-siRNA) in the balloon-injured carotid artery of hypercholesterolemic rabbits may lead to inhibition of MMP2 expression. Methods Two weeks after balloon injury, 5 µmol/l of Tamra-tagged MMP2-siRNA, scramble siRNA or saline was locally injected in the carotid artery and incubated for 1 h. Results Fluorescent microscopy studies showed the circumferential uptake of siRNA in the superficial layers of neointimal cells. MMP2 mRNA levels, measured by the real-time reverse transcriptase-polymerase chain reaction, was decreased by 79 ± 25% in MMP2-siRNA- versus scramble siRNA-transfected arteries (p < 0.05). MMP2 activity, measured by gelatin zymography performed on the conditioned media of MMP2-siRNA versus scramble siRNA transfected arteries, decreased by 53 ± 29%, 50 ± 24% and 46 ± 14% at 24, 48 and 72 h, respectively (p < 0.005 for all). No effect was observed on MMP9, pro-MMP9 and TIMP-2 levels. Conclusions The results obtained in the present study suggest that significant inhibition of gene expression can be achieved with local delivery of siRNA in the arterial wall in vivo. Copyright © 2008 John Wiley & Sons, Ltd. [source] Knockdown of Fc, receptor III in an arthritic temporomandibular joint reduces the nociceptive response in ratsARTHRITIS & RHEUMATISM, Issue 10 2010Phillip R. Kramer Objective Fc, receptor III (Fc,RIII; CD16) is a receptor expressed on immune cells that selectively binds IgG molecules. IgG binding results in cellular activation and cytokine release. IgG is an important factor in arthritis and can be found in the arthritic temporomandibular joint (TMJ). We undertook this study to test the hypothesis that a reduction in Fc,RIII expression in TMJ tissues would reduce the nociceptive and inflammatory responses in an inflamed joint. Methods Small interfering RNA (siRNA), either naked or complexed with linear polyethyleneimine, was injected into the superior joint space of the TMJ in rats. After administration of siRNA the joint was injected with saline or with Freund's complete adjuvant to induce arthritis. Nociceptive responses were quantitated in the rat by measuring the animal's meal duration. Fc,RIII expression in the TMJ tissue was assayed by immunocytochemistry or Western blotting. Cleavage of Fc,RIII transcript was then assayed by 5, rapid amplification of complementary DNA ends. Interleukin-1, (IL-1,) and IgG content was measured in the TMJ tissue by enzyme-linked immunosorbent assay. Results Injection of Fc,RIII siRNA reduced the amount of Fc,RIII in the TMJ tissues, and the transcript was cleaved in a manner consistent with an RNA interference mechanism. Moreover, injection of Fc,RIII siRNA reduced the nociceptive response of rats with an arthritic TMJ and reduced the amount of the proinflammatory cytokine IL-1,. Conclusion Fc,RIII contributes to the pain resulting from inflammatory arthritis of the TMJ, and siRNA has the potential to be an effective treatment for this disorder. [source] Glucocorticoids increase ,5 integrin expression and adhesion of synovial fibroblasts but inhibit ERK signaling, migration, and cartilage invasionARTHRITIS & RHEUMATISM, Issue 12 2009Torsten Lowin Objective In rheumatoid arthritis (RA), integrins mediate cell adhesion, migration, and invasion, and their expression is regulated by cytokines and growth factors. The aim of this study was to investigate whether hormones such as cortisol or other steroids can influence integrin expression and function in the synovial cells of patients with RA. Methods We performed immunofluorescence and fluorescence-activated cell sorting analyses to quantify surface integrin levels. Adhesion and migration assays were performed to study the function of synovial fibroblasts (SFs). ERK activation was measured by cellular activation of a signaling enzyme-linked immunosorbent assay. Invasion of SFs into cartilage was determined in the SCID mouse coimplantation model of RA in vivo. Results In RA, expression of integrin subunits ,5, ,v, and ,1 was higher at the site of invasion compared with the sublining zone. Testosterone and 17,-estradiol had no influence on integrin levels, but cortisol up-regulated expression of the ,5 subunit in a time-dependent and dose-dependent manner. In addition, cortisol increased the adhesion of SFs to fibronectin and inhibited ERK signaling upon integrin activation or upon stimulation with tumor necrosis factor. Small interfering RNA or a neutralizing antibody to ,5 integrin increased SF migration, indicating that up-regulated ,5 integrin is responsible for an immobile phenotype. In addition, in the SCID mouse model, SF invasion into cartilage was attenuated by glucocorticoid treatment in vivo. Conclusion Glucocorticoids increase integrin expression and the adhesion of cells to fibronectin, inhibit ERK signaling, and down-regulate the invasiveness of SFs in vivo. This study demonstrates that an important antiinflammatory aspect of glucocorticoids is regulating the expression and function of ,5 integrin. [source] TRAF6 knockdown promotes survival and inhibits inflammatory response to lipopolysaccharides in rat primary renal proximal tubule cellsACTA PHYSIOLOGICA, Issue 3 2010S. Liu Abstract Aim:, TRAF6 is a unique adaptor protein of the tumour necrosis factor receptor-associated factor family that mediates both tumour necrosis factor receptor (TNFR) and interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) signalling. Activation of IL-1R/TLR and TNFR pathways in renal tubular cells contributes to renal injury. This study aimed to investigate if blockade of lipopolysaccharide (LPS)-triggered TLR4 signalling by small interfering RNA (siRNA) targeting TRAF6 protects survival and inhibits inflammatory response in isolated rat renal proximal tubular cells (PTCs). Methods:, PTCs isolated from F344 rat kidneys were transfected with chemically synthesized siRNA targeting TRAF6 mRNA. Real-time quantitative PCR was applied to measure mRNA level of TRAF6, TNF-,, IL-6 and monocyte chemoattractant protein-1 (MCP-1). Protein levels of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase, caspase 3 and cleaved caspase 3 were evaluated by Western blotting. Cell viability was analysed with XTT reagents. Results:, We found that the TRAF6 gene was effectively silenced in PTCs using siRNA. TRAF6 knockdown resulted in reduced TNF-, and IL-6 mRNA expression upon LPS challenge. LPS-induced phosphorylation of JNK and p38 was attenuated in TRAF6 siRNA-transfected cells while the change in the phosphorylation of ERK was not remarkable. TRAF6 knockdown was associated with increased cell viability and reduced protein level of cleaved caspase-3, both, in the absence and presence of LPS. Conclusion:, Our studies suggest that TRAF6 knockdown may inhibit inflammatory response and promote cell survival upon LPS challenge in primary rat proximal renal tubular cells. [source] Delivery of small interfering RNA with a synthetic collagen poly(Pro-Hyp-Gly) for gene silencing in vitro and in vivoDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2010Taro Adachi Silencing gene expression by small interfering RNAs (siRNAs) has become a powerful tool for the genetic analysis of many animals. However, the rapid degradation of siRNA and the limited duration of its action in vivo have called for an efficient delivery technology. Here, we describe that siRNA complexed with a synthetic collagen poly(Pro-Hyp-Gly) (SYCOL) is resistant to nucleases and is efficiently transferred into cells in vitro and in vivo, thereby allowing long-term gene silencing in vivo. We found that the SYCOL-mediated local application of siRNA targeting myostatin, coding a negative regulator of skeletal muscle growth, in mouse skeletal muscles, caused a marked increase in the muscle mass within a few weeks after application. Furthermore, in vivo administration of an anti-luciferase siRNA/SYCOL complex partially reduced luciferase expression in xenografted tumors in vivo. These results indicate a SYCOL-based non-viral delivery method could be a reliable simple approach to knockdown gene expression by RNAi in vivo as well as in vitro. [source] Regulation of GADD153 induced by mechanical stress in cardiomyocytesEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2009W. P. Cheng Abstract Background, Growth arrest and DNA damage-inducible gene 153 (GADD153), an apoptosis regulated gene, increased during endoplasmic reticulum stress. However, the expression of GADD153 in cardiomyocytes under mechanical stress is little known. We aimed to investigate the regulation mechanism of GADD153 expression and apoptosis induced by mechanical stress in cardiomyocytes. Materials and methods, Aorta-caval shunt was performed in adult Sprague,Dawley rats to induce volume overload. Rat neonatal cardiomyocytes grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles min,1. Results, The increased ventricular dimension measured using echocardiography in the shunt group (n = 8) was reversed to normal by treatment with chaperon 4-phenylbutyric acid (PBA) (n = 8) at 500 mg kg,1 day,1 orally for 3 days. GADD153 protein and mRNA were up-regulated in the shunt group when compared with sham group (n = 8). Treatment with PBA reversed the protein of GADD153 to the baseline values. The TUNEL assay showed that PBA reduced the apoptosis induced by volume overload. Cyclic stretch significantly increased GADD153 protein and mRNA expression after 14 h of stretch. Addition of c-jun N-terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA and tumour necrosis factor-, (TNF-,) antibody 30 min before stretch, reduced the induction of GADD153 protein. Stretch increased, while GADD153-Mut plasmid, SP600125 and TNF-, antibody abolished the GADD153 promoter activity induced by stretch. GADD153 mediated apoptosis induced by stretch was reversed by GADD153 siRNA, GADD153-Mut plasmid and PBA. Conclusions, Mechanical stress enhanced apoptosis and GADD153 expression in cardiomyocytes. Treatment with PBA reversed both GADD153 expression and apoptosis induced by mechanical stress in cardiomyocytes. [source] Requirement of the tumour suppressor APC for the clustering of PSD-95 and AMPA receptors in hippocampal neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2007Atsushi Shimomura Abstract Mutations in the adenomatous polyposis coli (APC) gene are associated with familial adenomatous polyposis and sporadic colorectal tumours. The APC gene is expressed ubiquitously in various tissues, especially throughout the large intestine and central nervous system (CNS). In the CNS, the expression of the APC protein is highest during embryonic and early postnatal development. APC associates through its C-terminal region with postsynaptic density (PSD)-95, a neuronal protein that participates in synapse development. Here, we examined the involvement of APC in synaptogenesis. In cultured hippocampal neurons, both overexpression of a dominant-negative construct that disrupts the APC,PSD-95 interaction and knockdown of APC expression using small interfering RNA (siRNA) inhibited the clustering of PSD-95 and a glutamate receptor subunit, and reduced alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA)-induced activity of AMPA receptors; however, the clustering of an N -methyl- d -aspartate (NMDA) receptor subunit was unaffected. These results are suggestive of APC involvement in the development of glutamatergic synapses. [source] MyD88 expression in the rat dental follicle: implications for osteoclastogenesis and tooth eruptionEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 4 2010Dawen Liu Liu D, Yao S, Wise GE. MyD88 expression in the rat dental follicle: implications for osteoclastogenesis and tooth eruption. Eur J Oral Sci 2010; 118: 333,341. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci Myeloid differentiation factor 88 (MyD88) is a key adaptor molecule in the interleukin (IL)-1 and IL-18 toll-like receptor signaling pathways. Because MyD88 is present in dental follicle (DF) cells in vitro, the purpose of this study was to determine its chronological expression in vivo, as well as its possible role in osteoclastogenesis and tooth eruption. An oligo DNA microarray was used to determine expression of the Myd88 gene in vivo in the DFs from the first mandibular molars of postnatal rats from days 1 to 11. The results showed that MyD88 was expressed maximally on day 3. Using small interfering RNA (siRNA) to knock down MyD88 expression in the DF cells also reduced the expression of the nuclear factor-kappa B-1 (NFKB1) and monocyte chemoattractant protein 1 (MCP-1) genes. Interleukin-1, up-regulated the expression of NFKB1, MCP-1, and receptor activator of nuclear factor kappa B ligand (RANKL), but knockdown of MyD88 nullified this IL-1, effect. Conditioned medium from DF cells with MyD88 knocked down had reduced chemotactic activity for mononuclear cells and reduced osteoclastogenesis, as opposed to controls. In conclusion, the maximal expression of MyD88 in the DF of postnatal day 3 rats may contribute to the major burst of osteoclastogenesis needed for eruption by up-regulating MCP-1 and RANKL expression. [source] An estrogen receptor , suppressor, microRNA-22, is downregulated in estrogen receptor ,-positive human breast cancer cell lines and clinical samplesFEBS JOURNAL, Issue 7 2010Jianhua Xiong Previous studies have suggested that microRNAs (miRNAs) may play important roles in tumorigenesis, but little is known about the functions of most miRNAs in cancer development. In the present study, we set up a cell-based screen using a luciferase reporter plasmid carrying the whole , 4.7 kb 3,-UTR of estrogen receptor , (ER,) mRNA cotransfected with a synthetic miRNA expression library to identify potential ER,-targeting miRNAs. Among all the miRNAs, miR-22 was found to repress robustly the luciferase signal in both HEK-293T and ER,-positive MCF-7 cells. Mutation of the target site was found to abrogate this repression effect of miR-22, whereas antagonism of endogenous miR-22 in MDA-MB-231 cells resulted in elevated reporter signals. We assessed the miR-22 expression patterns in five breast cancer cell lines and 23 clinical biopsies and revealed that there is a significant inverse association between the miR-22 levels and ER, protein expression. To evaluate the potential of miR-22 as a potential therapeutic intervention, we found that reduction of endogenous ER, protein levels and suppression of cancer cell growth could be achieved in MCF-7 cells by miR-22 overexpression in a way that can be recapitulated by the introduction of specific small interfering RNA against ER,. The phenomena can be rescued by the reintroduction of ER,. Taken together, our data indicate that miR-22 was frequently downregulated in ER,-positive human breast cancer cell lines and clinical samples. Direct involvement in the regulation of ER, may be one of the mechanisms through which miR-22 could play a pivotal role in the pathogenesis of breast cancer. [source] Heme oxygenase-1/p21WAF1 mediates peroxisome proliferator-activated receptor-, signaling inhibition of proliferation of rat pulmonary artery smooth muscle cellsFEBS JOURNAL, Issue 6 2010Manxiang Li Activation of peroxisome proliferator-activated receptor (PPAR)-, suppresses proliferation of rat pulmonary artery smooth muscle cells (PASMCs), and therefore ameliorates the development of pulmonary hypertension in animal models. However, the molecular mechanisms underlying this effect remain largely unknown. This study addressed this issue. The PPAR, agonist rosiglitazone dose-dependently stimulated heme oxygenase (HO)-1 expression in PASMCs, 5 ,m rosiglitazone inducing a 12.1-fold increase in the HO-1 protein level. Cells pre-exposed to rosiglitazone showed a dose-dependent reduction in proliferation in response to serotonin; this was abolished by pretransfection of cells with sequence-specific small interfering RNA against HO-1. In addition, rosiglitazone stimulated p21WAF1 expression in PASMCs, a 2.34-fold increase in the p21WAF1 protein level being achieved with 5 ,m rosiglitazone; again, this effect was blocked by knockdown of HO-1. Like loss of HO-1, loss of p21WAF1 through siRNA transfection also reversed the inhibitory effect of rosiglitazone on PASMC proliferation triggered by serotonin. Taken together, our findings suggest that activation of PPAR, induces HO-1 expression, and that this in turn stimulates p21WAF1 expression to suppress PASMC proliferation. Our study also indicates that rosiglitazone, a medicine widely used in the treatment of type 2 diabetes mellitus, has potential benefits for patients with pulmonary hypertension. [source] Inhibition of PI3K/Akt partially leads to the inhibition of PrPC -induced drug resistance in gastric cancer cellsFEBS JOURNAL, Issue 3 2009Jie Liang Cellular prion protein (PrPC), a glycosyl-phosphatidylinositol-anchored membrane protein with unclear physiological function, was previous found to be upregulated in adriamycin (ADR)-resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901. Overexpression of PrPC in gastric cancer has certain effects on drug accumulation through upregulation of P-glycoprotein (P-gp), which is suggested to play an important role in determining the sensitivity of tumor cells to chemotherapy and is linked to activation of the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway. In the present study, we further investigate the role of the PI3K/Akt pathway in PrPC -induced multidrug-resistance (MDR) in gastric cancer. Immunohistochemistry and confocal microscope detection suggest a positive correlation between PrPC and phosphorylated Akt (p-Akt) expression in gastric cancer. Using established stable PrPC transfectant cell lines, we demonstrated that the level of p-Akt was increased in PrPC -transfected cells. Inhibition of PrPC expression by RNA interference resulted in decreased p-Akt expression. Inhibition of the PI3K/Akt pathway by one of its specific inhibitors, LY294002, or by Akt small interfering RNA (siRNA) resulted in decreased multidrug resistance of SGC7901 cells, partly through downregulation of P-gp induced by PrPC. Taken together, our results suggest that PrPC -induced MDR in gastric cancer is associated with activation of the PI3K/Akt pathway. Inhibition of PI3K/Akt by LY2940002 or Akt siRNA leads to inhibition of PrPC -induced drug resistance and P-gp upregulation in gastric cancer cells, indicating a possible novel mechanism by which PrPC regulates gastric cancer cell survival. [source] Gene expression silencing with ,specific' small interfering RNA goes beyond specificity , a study of key parameters to take into account in the onset of small interfering RNA off-target effectsFEBS JOURNAL, Issue 11 2008Sébastien Vankoningsloo RNA-mediated gene silencing (RNA interference) is a powerful way to knock down gene expression and has revolutionized the fields of cellular and molecular biology. Indeed, the transfection of cultured cells with small interfering RNAs (siRNAs) is currently considered to be the best and easiest approach to loss-of-function experiments. However, several recent studies underscore the off-target and potential cytotoxic effects of siRNAs, which can lead to the silencing of unintended mRNAs. In this study, we used a low-density microarray to assess gene expression modifications in response to five different siRNAs in various cell types and transfection conditions. We found major differences in off-target signature according to: (a) siRNA sequence; (b) cell type; (c) duration of transfection; and (d) post-transfection time before analysis. These results contribute to a better understanding of important parameters that could impact on siRNA side effects in knockdown experiments. [source] Design of Multiresponsive Hydrogel Particles and AssembliesADVANCED FUNCTIONAL MATERIALS, Issue 11 2010Grant R. Hendrickson Abstract In the realm of soft nanotechnology, hydrogel micro- and nanoparticles represent a versatile class of responsive materials. Over the last decade, our group has investigated the synthesis and physicochemical properties of a variety of synthetic hydrogel particles. From these efforts, several particle types have emerged with potentially enabling features for biological applications, including nanogels for targeted drug delivery, microlenses for biosensing, and coatings for biomedical devices. For example, core/shell nanogels have been used to encapsulate and deliver small interfering RNA to ovarian cancer cells; nanogels used in this fashion may improve therapeutic outcomes for a variety of macromolecular therapeutics. Microgels arranged as multilayers on implantable biomaterials greatly minimize the host inflammatory response to the material. Furthermore, the triggered release of drugs (i.e., insulin) has been demonstrated from similar assemblies. The goal of this feature article is to highlight developments in the design of responsive microgels and nanogels in the context of our recent efforts and in relation to the community that has grown up around this fascinating class of materials. [source] Lipid-Like Nanoparticles for Small Interfering RNA Delivery to Endothelial CellsADVANCED FUNCTIONAL MATERIALS, Issue 19 2009Seung-Woo Cho Abstract Here, nanoparticles composed of lipid-like materials (lipidoids) to facilitate non-viral delivery of small interfering RNA (siRNA) to endothelial cells (ECs) are developed. Nanoparticles composed of siRNA and lipidoids with small size (,200,nm) and positive charge (,34,mV) are formed by self-assembly of lipidoids and siRNA. Ten lipidoids are synthesized and screened for their ability to facilitate the delivery of siRNA into ECs. Particles composed of leading lipidoids show significantly better delivery to ECs than a leading commercially available transfection reagent, Lipofectamine 2000. As a model of potential therapeutic application, nanoparticles composed of the top performing lipidoid, NA114, are studied for their ability to deliver siRNA targeting anti-angiogenic factor (SHP-1) to human ECs. Silencing of SHP-1 expression significantly enhances EC proliferation and decreases EC apoptosis under a simulated ischemic condition. [source] ERK5 is a target for gene amplification at 17p11 and promotes cell growth in hepatocellular carcinoma by regulating mitotic entryGENES, CHROMOSOMES AND CANCER, Issue 2 2009Keika Zen Using high-density oligonucleotide microarrays, we investigated DNA copy-number aberrations in cell lines derived from hepatocellular carcinomas (HCCs) and detected a novel amplification at 17p11. To identify the target of amplification at 17p11, we defined the extent of the amplicon and examined HCC cell lines for expression of all seven genes in the 750-kb commonly amplified region. Mitogen-activated protein kinase (MAPK) 7, which encodes extracellular-regulated protein kinase (ERK) 5, was overexpressed in cell lines in which the gene was amplified. An increase in MAPK7 copy number was detected in 35 of 66 primary HCC tumors. Downregulation of MAPK7 by small interfering RNA suppressed the growth of SNU449 cells, the HCC cell line with the greatest amplification and overexpression of MAPK7. ERK5, phosphorylated during the G2/M phases of the cell cycle, regulated entry into mitosis in SNU449 cells. In conclusion, our results suggest that MAPK7 is likely the target of 17p11 amplification and that the ERK5 protein product of MAPK7 promotes the growth of HCC cells by regulating mitotic entry. © 2008 Wiley-Liss, Inc. [source] Biodegradable Dextran Nanogels for RNA Interference: Focusing on Endosomal Escape and Intracellular siRNA DeliveryADVANCED FUNCTIONAL MATERIALS, Issue 9 2009Koen Raemdonck Abstract The successful therapeutic application of small interfering RNA (siRNA) largely relies on the development of safe and effective delivery systems that are able to guide the siRNA therapeutics to the cytoplasm of the target cell. In this report, biodegradable cationic dextran nanogels are engineered by inverse emulsion photopolymerization and their potential as siRNA carriers is evaluated. The nanogels are able to entrap siRNA with a high loading capacity, based on electrostatic interaction. Confocal microscopy and flow cytometry analysis reveal that large amounts of siRNA-loaded nanogels can be internalized by HuH-7 human hepatoma cells without significant cytotoxicity. Following their cellular uptake, it is found that the nanogels are mainly trafficked towards the endolysosomes. The influence of two different strategies to enhance endosomal escape on the extent of gene silencing is investigated. It is found that both the application of photochemical internalization (PCI) and the use of an influenza-derived fusogenic peptide (diINF-7) can significantly improve the silencing efficiency of siRNA-loaded nanogels. Furthermore, it is shown that an efficient gene silencing requires the degradation of the nanogels. As the degradation kinetics of the nanogels can easily be tailored, these particles show potential for intracellular controlled release of short interfering RNA. [source] Cytochrome c oxidase isoform IV-2 is involved in 3-nitropropionic acid-induced toxicity in striatal astrocytesGLIA, Issue 14 2009Shilpee Singh Abstract Astrocyte mitochondria play an important role for energy supply and neuronal survival in the brain. Toxic and degenerative processes are largely associated with mitochondrial dysfunction. We, therefore, investigated the effect of 3-nitropropionic acid (NPA), a mitochondrial toxin and in vitro model of Huntington's disease (HD), on mitochondrial function and viability of primary striatal astrocytes. Although NPA is known as an irreversible inhibitor of succinate dehydrogenase, we observed an increase of astrocyte ATP levels after NPA treatment. This effect could be explained by NPA-mediated alterations of cytochrome c oxidase subunit IV isoform (COX IV) expression. The up-regulation of COX isoform IV-2 caused an increased enzyme activity at the expense of elevated mitochondrial peroxide production causing increased cell death. The application of a small interfering RNA against COX IV-2 revealed the causal implication of COX isoform IV-2 in NPA-mediated elevation of oxidative stress and necrotic cell death. Thus, we propose a novel, additional mechanism of NPA-induced cell stress and death which is based on structural and functional changes of astrocyte COX and which could indirectly impair neuronal survival. © 2009 Wiley-Liss, Inc. [source] Inhibition of poly adenosine diphosphate-ribose polymerase decreases hepatocellular carcinoma growth by modulation of tumor-related gene expression,HEPATOLOGY, Issue 1 2010Rosa Quiles-Perez Hepatocellular carcinoma (HCC) is associated with a poor prognosis due to a lack of effective treatment options. In HCC a significant role is played by DNA damage and the inflammatory response. Poly (ADP-ribose) polymerase-1 (PARP-1) is an important protein that regulates both these mechanisms. The objective of this study was to examine the effect of pharmacology PARP-1 inhibition on the reduction of tumor volume of HCC xenograft and on the hepatocarcinogenesis induced by diethyl-nitrosamine (DEN). Pharmacologic PARP-1 inhibition with DPQ greatly reduces tumor xenograft volume with regard to a nontreated xenograft (394 mm3 versus 2,942 mm3, P < 0.05). This observation was paralleled by reductions in xenograft mitosis (P = 0.02) and tumor vasculogenesis (P = 0.007, confirmed by in vitro angiogenesis study), as well as by an increase in the number of apoptotic cells in DPQ-treated mice (P = 0.04). A substantial difference in key tumor-related gene expression (transformed 3T3 cell double minute 2 [MDM2], FLT1 [vascular endothelial growth factor receptor-1, VEGFR1], epidermal growth factor receptor [EPAS1]/hypoxia-inducible factor 2 [HIF2A], EGLN1 [PHD2], epidermal growth factor receptor [EGFR], MYC, JUND, SPP1 [OPN], hepatocyte growth factor [HGF]) was found between the control tumor xenografts and the PARP inhibitor-treated xenografts (data confirmed in HCC cell lines using PARP inhibitors and PARP-1 small interfering RNA [siRNA]). Furthermore, the results obtained in mice treated with DEN to induce hepatocarcinogenesis showed, after treatment with a PARP inhibitor (DPQ), a significant reduction both in preneoplastic foci and in the expression of preneoplastic markers and proinflammatory genes (Gstm3, Vegf, Spp1 [Opn], IL6, IL1b, and Tnf), bromodeoxyuridine incorporation, and NF-,B activation in the initial steps of carcinogenesis (P < 0.05). Conclusion: This study shows that PARP inhibition is capable of controlling HCC growth and preventing tumor vasculogenesis by regulating the activation of different genes involved in tumor progression. (HEPATOLOGY 2010;51:255,266.) [source] Cyclin-dependent kinase 1 plays a critical role in DNA replication control during rat liver regeneration,HEPATOLOGY, Issue 6 2009Delphine Garnier Liver regeneration is a unique process to restore hepatic homeostasis through rapid and synchronous proliferation of differentiated hepatocytes. Previous studies have shown that hepatocyte proliferation is characterized by high expression levels of the "mitotic" cyclin-dependent kinase 1 (Cdk1) during S-phase compared to other mammalian cells. In the light of findings showing that Cdk1 compensates for the loss of Cdk2 and drives S-phase in Cdk2-deficient cells derived from Cdk2 knockout mice, we took advantage of the models of liver regeneration following partial hepatectomy and primary cultures of normal rat hepatocytes to further examine the involvement of Cdk1 during DNA replication in hepatocytes and to dissect specific cell cycle regulation in hepatocytes compared to control human foreskin fibroblasts. In hepatocytes, Cdk1 exhibited a biphasic activation pattern correlating S-phase and G2/M transition, bound to cyclin A or B1 and localized to the nucleus during DNA replication. Importantly, small interfering RNA (siRNA)-mediated silencing of Cdk1 led to a strong decrease in DNA synthesis without affecting centrosome duplication. Furthermore, in hepatocytes arrested by the iron chelator O-Trensox in early S-phase prior to DNA replication, Cdk1/cyclin complexes were active, while replication initiation components such as the minichromosome maintenance 7 (Mcm7) protein were loaded onto DNA. Moreover, Mcm7 expression and loading onto DNA were not modified by Cdk1 silencing. Conversely, in fibroblasts, Cdk1 expression and activation were low in S-phase and its silencing did not reduce DNA synthesis. Conclusion: Cdk1 is essential for DNA replication downstream formation of replication initiation complexes in hepatocytes but not in fibroblasts and, as such, our data exemplify crucial differences in the cell cycle regulation between various mammalian cell types. (HEPATOLOGY 2009.) [source] Hepatitis C virus replication is inhibited by 22,-methoxyolean-12-ene-3,, 24(4,)-diol (ME3738) through enhancing interferon-,,HEPATOLOGY, Issue 1 2008Yoichi Hiasa A derivative of soyasapogenol, 22,-methoxyolean-12-ene-3,, 24(4,)-diol (ME3738), ameliorates liver injury induced by Concanavalin A in mice. We examined whether ME3738 has independent antiviral effects against hepatitis C virus (HCV) using an established HCV replication model that expresses the full-length genotype 1a HCV complementary DNA plasmid (pT7-flHCV-Rz) under the control of a replication-defective adenoviral vector expressing T7 polymerase. Hepatocellular carcinoma (HepG2) cells, human hepatoma (Huh7) cells, or monkey kidney (CV-1) cells were transfected with pT7-flHCV-Rz, and infected with adenoviral vector expressing T7 polymerase. ME3738 or interferon-, (IFN-,) was added thereafter and then protein and RNA were harvested from the cells at 9 days after infection. HCV-positive and HCV-negative strands were measured by real-time reverse-transcription polymerase chain reaction and HCV core protein expression was measured using an enzyme-linked immunosorbent assay. The messenger RNA levels of innate antiviral response-related genes were assessed using real-time reverse-transcription polymerase chain reaction. ME3738 dose-dependently reduced HCV-RNA and core protein in hepatocyte-derived cell lines. The antiviral effect was more pronounced in HepG2 than in Huh7 cells. ME3738 increased messenger RNA levels of interferon-, (IFN-,) and of IFN-stimulated genes (2,-5, oligoadenylate synthetase, myxovirus resistance protein A [MxA]). Interferon-, knockdown by small interfering RNA abrogated the anti-HCV effect of ME3738. Moreover, the anti-HCV effects were synergistic when ME3738 was combined with IFN-,. Conclusion: ME3738 has antiviral effects against HCV. The enhancement of autocrine IFN-, suggests that ME3738 exerts antiviral action along the type I IFN pathway. This anti-HCV action by ME3738 was synergistically enhanced when combined with IFN-,. ME3738 might be a useful anti-HCV drug either with or without IFN-,. (HEPATOLOGY 2008.) [source] Mechanisms of protection by the betaine-homocysteine methyltransferase/betaine system in HepG2 cells and primary mouse hepatocytes,HEPATOLOGY, Issue 5 2007Cheng Ji Betaine-homocysteine methyltransferase (BHMT) regulates homocysteine levels in the liver. We previously reported that the alteration of BHMT is associated with alcoholic liver steatosis and injury. In this study, we tested whether BHMT protects hepatocytes from homocysteine-induced injury and lipid accumulation. Both BHMT transfectants of HepG2 cells and primary mouse hepatocytes with suppressed BHMT were generated. Comparisons were made between the cell models with respect to their response to homocysteine treatments. Homocysteine metabolism was impaired in HepG2 cells, and the expression of BHMT in HepG2 cells ameliorated the impairment and stabilized the levels of intracellular homocysteine after the addition of exogenous homocysteine. BHMT expression inhibited homocysteine-induced glucose-regulated protein 78 (GRP78) and C/EBP-homologous protein (CHOP) and homocysteine-induced cell death. A betaine treatment protected primary mouse hepatocytes from a homocysteine-induced increase in GRP78 and cell death but not a tunicamycin-induced increase. Homocysteine induced greater CHOP expression (2.7-fold) in BHMT small interfering RNA (siRNA),transfected cells than in a control (1.9-fold). Homocysteine-induced cell death was increased by 40% in the siRNA-treated cells in comparison with the control. Apolipoprotein B (apoB) expression was higher and triglycerides and cholesterol were lower in HepG2 expressing BHMT. In primary mouse hepatocytes, homocysteine induced the accumulation of triglycerides and cholesterol, which was reduced in the presence of betaine. Betaine partially reduced homocysteine-induced sterol regulatory element binding protein 1 expression in HepG2 cells and increased S-adenosylmethionine in primary mouse hepatocytes. Conclusion: The BHMT/betaine system directly protects hepatocytes from homocysteine-induced injury but not tunicamycin-induced injury, including an endoplasmic reticulum stress response, lipid accumulation, and cell death. This system also exhibits a more generalized effect on liver lipids by inducing ApoB expression and increasing S-adenosylmethionine/S-adenosylhomocysteine. (HEPATOLOGY 2007.) [source] Silencing MAT2A gene by RNA interference inhibited cell growth and induced apoptosis in human hepatoma cellsHEPATOLOGY RESEARCH, Issue 5 2007Quanyan Liu Aims:, A switch in gene expression from MAT1A to MAT2A was found in liver cancer, suggesting that MAT2A plays an important role in facilitating cancer growth. MAT2A is an interesting target for antineoplastic therapy. The molecular mechanisms of silencing MAT2A by RNA interference inhibited cell growth and induced apoptosis in hepatoma cells was studied. Methods:, We investigated the effects of MAT2A on S-adenosyl-methionine (SAM) production, cell growth and apoptotic cell death in hepatoma cell lines (Bel-7402, HepG2, and Hep3B) using an RNA interference approach. Results:, The treatment of three hepatoma cell lines with small interfering RNA (siRNA) targeting to the MAT2A gene resulted in reducing the MAT II activity, facilitating SAM production, increasing SAM : SAH ratio, inhibiting cell growth and inducing cell apoptosis in hepatoma cells. In addition, silencing MAT2A gene resulted in the stimulation of MAT1A mRNA production, which was blocked by 3-deazaadenosine and l -ethionine, but not d -ethionine, suggesting that such effect was specific and mediated by upregulation of SAM level and SAM : S-adenosylethionine (SAH) ratio. Conclusion:, Silencing MAT2A by sequence-specific small interfering RNA caused a switch of MAT gene expression from MAT2A to MAT1A, which led the content of SAM to change to a higher steady-state level that resulted in the inhibition of cell growth and the induction of apoptotic cell death in human hepatoma cells. These results also suggested that MAT2A may hold potential as a new target for liver cancer gene therapy. [source] Expression of estrogen receptor alpha increases leptin-induced STAT3 activity in breast cancer cellsINTERNATIONAL JOURNAL OF CANCER, Issue 1 2010Nadine A. Binai Abstract Adipositas correlates with an enhanced risk of developing malignant diseases such as breast cancer, endometrial tumor or prostate carcinoma, but the molecular basis for this is not well understood. Potential mechanisms include increased bioavailability of adipocytokines (e.g. leptin) and steroid hormones. Here, we investigated cross-talk between ER, (estrogen receptor alpha) and leptin-induced activation of signal transducer and activator of transcription 3 (STAT3), a transactivator of important oncogenes. Upon leptin binding to its receptor Ob-RL (obesity receptor), STAT3 tyrosine phosphorylation and transactivation activity were enhanced by simultaneously expressing ER,. Downregulation of ER, using small interfering RNA abolished leptin-induced STAT3 phosphorylation. Interestingly, leptin-mediated STAT3 activation was unaffected by co-stimulation with the ER, ligands estradiol (E2) or estrogen antagonists ICI182,780 and tamoxifen, implying that enhancement of leptin-mediated STAT3 activity is independent of ER, ligands. We also detected ER, binding to STAT3 and JAK2 (Janus kinase 2), resulting in enhanced JAK2 activity upstream of STAT3 in response to leptin that might lead to an increased ER,-dependent cell viability. Altogether, our results indicate that leptin-induced STAT3 activation acts as a key event in ER,-dependent development of malignant diseases. [source] CXCR7 is inducible by HTLV-1 Tax and promotes growth and survival of HTLV-1-infected T cellsINTERNATIONAL JOURNAL OF CANCER, Issue 9 2009Zhe Jin Abstract Human T-lymphotropic virus type 1 (HTLV-1), the etiological agent of adult T-cell leukemia (ATL), encodes the potent transcriptional activator Tax, which is required for HTLV-1-induced immortalization of T cells. CXCR7 is an atypical chemokine receptor frequently expressed by tumor cells and known to promote cell growth and survival. We found that HTLV-1-immortalized T cells expressing Tax consistently expressed CXCR7. Induction of Tax in JPX-9 upregulated CXCR7. Wild-type Tax efficiently activated the CXCR7 promoter via a proximal NF-,B site, while a mutant Tax selectively defective in NF-,B activation did not. CCX754, a synthetic CXCR7 antagonist, inhibited cell growth and increased apoptosis of HTLV-1-immortalized T cells. Knockdown of CXCR7 by small interfering RNA also reduced cell growth. Stable expression of CXCR7 in a CXCR7-negative ATL cell line promoted cell growth and survival. Taken together, CXCR7 is inducible by Tax and may play an important role in HTLV-1-induced immortalization of T cells by promoting growth and survival of HTLV-1-infected T cells. © 2009 UICC [source] | ||||||||||||||||||||||||||||||||||