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Results for “Small interfering RNA”

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At least 217 records · Page 12Linked to original sources

PubMed2003

Caveolin-1 knockdown by small interfering RNA suppresses responses to the chemokine monocyte chemoattractant protein-1 by human astrocytes.

Astrocytes regulate the integrity of the blood-brain barrier and influence inflammatory processes in the central nervous system. The pro-inflammatory chemokine monocyte chemoattractant protein-1 (MCP-1), which is both released by and stimulates astrocytes, is thought to play a crucial role in both these activities. Because astrocytes have been shown to possess caveolae, vesicular structures that participate in intracellular transport and signal transduction events, we reasoned that expression of the major structural protein of these organelles, caveolin-1, might feature critically in the cellular responses to MCP-1. To test this hypothesis, caveolin-1 level was "knocked down" in human astrocyte cultures by using a small interfering RNA approach. This method resulted in efficient (>90% loss) and specific knockdown of caveolin-1 expression while sparring glial fibrillary acidic protein as well as several other proteins involved in endocytosis. Astrocytes suffering caveolin-1 loss showed diminished ability to down-modulate and internalize the MCP-1 receptor (CCR2) in response to exposure to this chemokine and also demonstrated significantly reduced capacity to undergo chemotaxis and calcium flux when MCP-1-stimulated. The results highlight a potentially prominent role for caveolae and/or caveolin-1 in mediating astrocyte responses to MCP-1, a feature that might significantly dictate the progression of inflammatory events at the blood-brain barrier.

Astrocytes↗

PubMed2005

Down-regulation of Cx43 by retroviral delivery of small interfering RNA promotes an aggressive breast cancer cell phenotype.

Connexins are gap junction proteins that assemble into channels that mediate direct intercellular communication. Connexins are well-documented tumor suppressors and are thought to regulate both cell growth and differentiation. As previously reported, most human breast tumors and cell lines down-regulate gap junctions or have defective gap junctional intercellular communication. Furthermore, overexpression of connexins in breast cancer cells inhibits tumor growth in vivo. In this study, we hypothesize that controlled Cx43 down-regulation would induce breast tumor cells to acquire a more aggressive phenotype. Here we report that Cx43 was down-regulated in both normal rat kidney (NRK) cells and human breast cancer cell lines (MDA-MB-231 and Hs578T) by transfection with chemically synthesized small interfering RNA (siRNA) or short hairpin RNA generated from a retroviral infection. Furthermore, we show that retroviral delivery and expression of siRNA directed to different coding regions of Cx43 resulted in differential levels of Cx43 silencing and impaired gap junctional intercellular communication. Cx43-silenced Hs578T cells grew faster and were more migratory. Finally, Western blot analysis revealed that down-regulation of Cx43 resulted in decreased expression of thrombospondin-1, an antiangiogenesis molecule, and increased expression of vascular endothelial growth factor. Taken together, these results suggest that Cx43 is required for maintaining cell differentiation and the regulation of molecules important in angiogenesis.

Animals↗

PubMed2006

Adenovirus-mediated small interfering RNA against matrix metalloproteinase-2 suppresses tumor growth and lung metastasis in mice.

Matrix metalloproteinases (MMP) are a group of proteinases that have normal physiologic roles degrading and remodeling the extracellular matrix. They also have multiple roles in different stages of tumor progression. Elevated levels of MMPs have been observed in many tumors; these increases have a strong association with the invasive phenotype. MMP-2 and MMP-9 are particularly involved in cancer invasion and metastasis. MMP inhibitors are currently being tested as therapeutic agents for a number of cancers in both preclinical models and in clinical trials. To date, clinical trials using this strategy have had limited efficacy. A major concern is the lack of specificity of commercially available MMP inhibitors. An adenoviral vector expressing small interfering RNA against the MMP-2 gene (Ad-MMP-2) was constructed to specifically inhibit MMP-2 expression. The effect of Ad-MMP-2 on invasion, angiogenesis, tumor growth, and metastasis of A549 lung cancer cell was evaluated. Ad-MMP-2 infection of lung cancer cells showed specific down-regulation of MMP-2 protein, activity, and transcription as determined by Western blotting, gelatin zymography, and reverse transcription-PCR. Ad-MMP-2 inhibition also mitigated lung cancer invasion and migration, and reduced tumor cell-induced angiogenesis in vitro. In an experimental metastatic lung tumor model, treatment of established tumors by Ad-MMP-2 inhibited s.c. tumor growth and formation of lung nodules in mice. Adenoviral-mediated RNA interference against MMP-2 has significant therapeutic potential for lung cancer and exerts some of this effect by inhibiting angiogenesis.

Adenoviridae↗

PubMed2005

Knockdown of c-Met by adenovirus-delivered small interfering RNA inhibits hepatocellular carcinoma growth in vitro and in vivo.

c-Met is highly expressed and constitutively activated in various human tumors. We employed adenovirus-mediated RNA interference technique to knock down c-Met expression in hepatocellular carcinoma cells and observed its effects on hepatocellular carcinoma cell growth in vitro and in vivo. Among the five hepatocellular carcinoma and one normal human liver cell lines we analyzed, c-Met was highly expressed and constitutively tyrosine phosphorylated in only MHCC97-L and HCCLM3 hepatocellular carcinoma cells. Knockdown of c-Met could inhibit MHCC97-L cells proliferation by arresting cells at G0-G1 phase. Soft agar colony formation assay indicated that the colony forming ability of MHCC97-L cells decreased by approximately 70% after adenovirus AdH1-small interfering RNA (siRNA)/met infection. In vivo experiments showed that adenovirus AdH1-siRNA/met inhibited the tumorigenicity of MHCC97-L cells and significantly suppressed tumor growth when injected directly into tumors. These results suggest that knockdown of c-Met by adenovirus-delivered siRNA may be a potential therapeutic strategy for treatment of hepatocellular carcinoma in which c-Met is overexpressed.

Adenoviridae↗

PubMed2005

Small interfering RNA inhibition of SPARC attenuates the profibrotic effect of transforming growth factor beta1 in cultured normal human fibroblasts.

OBJECTIVE: SPARC (secreted protein, acidic and rich in cysteine), or osteonectin, is a matricellular protein. Recently, it was observed to be overexpressed in fibroblasts obtained from the skin of patients with scleroderma, as well as in different tissues from patients with several other fibrotic disorders. Moreover, a genetic polymorphism in SPARC has been associated with susceptibility to scleroderma. Transforming growth factor beta1 (TGFbeta1) is a profibrotic cytokine that stimulates excessive collagen production in patients with scleroderma or other fibrotic diseases. The purpose of this study was to examine whether specific inhibition of SPARC can influence the expression of type I collagen and ameliorate the profibrotic activity of TGFbeta1 on normal human fibroblasts. METHODS: Fibroblasts obtained from the skin of 4 healthy individuals were cultured and transfected with SPARC small interfering RNA (siRNA). TGFbeta was used as a fibrosis stimulus in cultured fibroblasts. Real-time quantitative reverse transcriptase-polymerase chain reaction and Western blotting were used to measure transcription and protein levels of SPARC and type I collagen, respectively. RESULTS: The fibroblasts transfected with SPARC siRNA showed decreased expression of both SPARC and type I collagen. Exogenous TGFbeta1 induced increased expression of both SPARC and type I collagen in cultured normal human fibroblasts, but this response was significantly blunted in the fibroblasts transfected with SPARC siRNA. CONCLUSION: TGFbeta1 can induce increased expression of both SPARC and type I collagen. Specific inhibition of SPARC led to decreased expression of type I collagen and attenuated the profibrotic effect of TGFbeta1 in cultured normal human fibroblasts. Use of siRNA to silence SPARC represents a potential therapeutic approach to fibrotic disorders such as scleroderma.

Cells, Cultured↗

PubMed2006

Silencing mammalian target of rapamycin signaling by small interfering RNA enhances rapamycin-induced autophagy in malignant glioma cells.

The mammalian target of rapamycin (mTOR) plays a central role in regulating the proliferation of malignant glioma cells, and mTOR-specific inhibitors such as rapamycin analogs are considered as promising therapy for malignant gliomas. However, the efficacy of mTOR inhibitors alone in the treatment of patients with malignant gliomas is only modest, potentially because these agents rather than acting as mTOR kinase inhibitors instead interfere with the function of only mTOR/raptor (regulatory-associated protein of mTOR) complex and thus do not perturb all mTOR functions. The purpose of this study was to determine whether global inhibition of the mTOR molecule enhances the antitumor effect of rapamycin on malignant glioma cells. We showed that rapamycin induced autophagy and that inhibition of autophagy by small interfering RNA (siRNA) directed against autophagy-related gene Beclin 1 attenuated the cytotoxicity of rapamycin in rapamycin-sensitive tumor cells, indicating that the autophagy was a primary mediator of rapamycin's antitumor effect rather than a protective response. Exogenous expression of an mTOR mutant interfering with its kinase activity markedly enhanced the incidence of rapamycin-induced autophagy. Moreover, silencing of mTOR with siRNA augmented the inhibitory effect of rapamycin on tumor cell viability by stimulating autophagy. Importantly, not only rapamycin-sensitive malignant glioma cells with PTEN mutations but also rapamycin-resistant malignant glioma cells with wild-type PTEN were sensitized to rapamycin by mTOR siRNA. These results indicate that rapamycin-induced autophagy is one of the agent's antitumor effects and that silencing or inhibiting mTOR kinase activity could enhance the effectiveness of rapamycin.

Autophagy↗

PubMed2005

Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and gamma radiation.

Ku protein is a heterodimer (Ku70 and Ku86) known to play an important role in V(D)J recombination, apoptosis, telomere fusion, and double-strand break repair. Its role in double-strand breaks is relevant to cancer therapy because lack of Ku86 causes one of the most radiation-responsive phenotypes (hamster cells, XRS5). Although it is known that the heterodimer is necessary for the various functions of this protein, the impact of targeting Ku in human cancer cells has not been shown due to lack of appropriate approaches. It is also not known whether complete knock-out of Ku protein is required to enhance the sensitivity of human cells to gamma radiation as Ku protein is much more abundant in human cells than in hamster cells. In the current article, we have investigated the direct effect of Ku70 depletion in human cervical epithelioid (HeLa) and colon carcinoma (HCT116) cells. We specifically targeted Ku70 mRNA by use of small interfering RNA (siRNA). Of the five Ku70 siRNA synthesized, three inhibited the expression of Ku70 by up to 70% in HeLa cells. We have tested the effect of chemically synthesized siRNAs for target sequence 5 (CS #5) on the response of HeLa cells 72 hours after transfection to gamma radiation and etoposide, as this showed the maximum inhibition of Ku70 expression. Ku70 siRNA induced a decrease in the surviving fraction of irradiated HeLa cells by severalfold. Similar sensitizing effects were observed for etoposide, a topoisomerase II inhibitor. Studies with HCT116 cells using the same Ku70 siRNA (CS #5) showed a direct correlation between expression of Ku70 and sensitization to radiation and etoposide treatments.

Animals↗

PubMed2005

Modulation of gene expression by antisense and antigene oligodeoxynucleotides and small interfering RNA.

Antisense oligodeoxynucleotides, triplex-forming oligodeoxynucleotides and double-stranded small interfering RNAs have great potential for the treatment of many severe and debilitating diseases. Concerted efforts from both industry and academia have made significant progress in turning these nucleic acid drugs into therapeutics, and there is already one FDA-approved antisense drug in the clinic. Despite the success of one product and several other ongoing clinical trials, challenges still exist in their stability, cellular uptake, disposition, site-specific delivery and therapeutic efficacy. The principles, strategies and delivery consideration of these nucleic acids are reviewed. Furthermore, the ways to overcome the biological barriers are also discussed so that therapeutic concentrations at their target sites can be maintained for a desired period.

Animals↗

PubMed2004

Identification of cytoskeletal [14C]carboplatin-binding proteins reveals reduced expression and disorganization of actin and filamin in cisplatin-resistant cell lines.

Cisplatin resistant (CP-r) cells often show decreased uptake of cisplatin in association with reduced cell surface proteins and decreased endocytosis. In this report, two major [14C]carboplatin-binding proteins were identified as filamin and actin by photoaffinity labeling and mass spectrometry. Decreased expression of these two proteins was found in two different human CP-r cell lines (KB-CP20 and 7404-CP20), in comparison with their parental cell lines (KB-3-1 and BEL-7404), respectively. Disorganization of beta-actin and filamin 250 and 90 was also detected in these CP-r cells by confocal microscopy. Transfection of a wild-type actin-enhanced green fluorescent protein (EGFP) expression vector into 7404-CP20 cells resulted in a nonfilamentous actin-EGFP distribution compared with a normal distribution in the cisplatin-sensitive BEL-7404 cells, suggesting that cytoskeletal organization is disturbed in the CP-r cells. The identification of actin and filamin as [14C]carboplatin-binding proteins and decreased expression and disorganization of several cytoskeletal proteins in CP-r cells provide a molecular and cellular basis for the known defect in endocytosis in these cells.

Actins↗

PubMed2006

Improving RNA interference in mammalian cells by 4'-thio-modified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2'-O-alkyl modifications.

A systematic structure-activity relationship study of 4'-thioribose containing small interfering RNAs (siRNAs) has led to the identification of highly potent and stable antisense constructs. To enable this optimization effort for both in vitro and in vivo applications, we have significantly improved the yields of 4'-thioribonucleosides by using a chirally pure (R)-sulfoxide precursor. siRNA duplexes containing strategically placed regions of 4'-thio-RNA were synthesized and evaluated for RNA interference activity and plasma stability. Stretches of 4'-thio-RNA were well tolerated in both the antisense and sense strands. However, optimization of both the number and placement of 4'-thioribonucleosides was necessary for maximal potency. These optimized siRNAs were generally equipotent or superior to native siRNAs and exhibited increased thermal and plasma stability. Furthermore, significant improvements in siRNA activity and plasma stability were achieved by judicious combination of 4'-thioribose with 2'-O-methyl and 2'-O-methoxyethyl modifications. These optimized 4'-thio-siRNAs may be valuable for developing stable siRNAs for therapeutic applications.

Animals↗

PubMed2004

Silencing expression of the clusterin/apolipoprotein j gene in human cancer cells using small interfering RNA induces spontaneous apoptosis, reduced growth ability, and cell sensitization to genotoxic and oxidative stress.

Clusterin/Apolipoprotein J (CLU) is a heterodimeric ubiquitously expressed secreted glycoprotein that is implicated in several physiological processes and is differentially expressed in many severe physiological disturbances, including tumor formation and in vivo cancer progression. Despite extensive efforts, clarification of CLU's biological role has been exceptionally difficult and its precise function remains elusive. Short RNA duplexes, referred to as small interfering RNAs (siRNAs), provide a new approach for the elucidation of gene function in human cells. Here, we describe siRNA-mediated CLU gene silencing in osteosarcoma and prostate human cancer cells and illustrate that CLU mRNA is amenable to siRNA-mediated degradation. Our data demonstrate that CLU knockdown in human cancer cells induces significant reduction of cellular growth and higher rates of spontaneous endogenous apoptosis. Moreover, CLU knockdown cancer cells were significantly sensitized to both genotoxic and oxidative stress induced by chemotherapeutic drugs and H(2)O(2), respectively. These effects were more pronounced in cell lines that express high endogenous steady-state levels of the CLU protein and occur through hyperactivation of the cellular apoptotic machinery. Overall, our results reveal that, in the distinct cellular contexts of the osteosarcoma and prostate cancer cells assayed, CLU is a central molecule in cell homeostasis that exerts a cytoprotective function. The described CLU-specific siRNA oligonucleotides that can potently silence CLU gene expression may thus prove valuable agents during antitumor therapy or at other pathological conditions where CLU has been implicated.

Apoptosis↗

PubMed2006

Small interfering RNA (siRNA) delivery into monocyte-derived dendritic cells by electroporation.

Selective gene silencing by small interfering RNAs (siRNAs) has been shown to be an efficient method for the targeted manipulation of cellular functions. Chemical transfection reagents represent the current standard technique in siRNA duplex delivery into mammalian cells. However, when trying to manipulate cells isolated from patients in clinical approaches, chemical agents might cause unwanted side effects, such as allergic reactions, or interfere with other cellular functions. In this study we describe electroporation as a suitable and efficient method for the delivery of siRNA into monocyte-derived dendritic cells (moDCs). Using a fluorescein-labeled non-silencing siRNA duplex as a model system, we carefully investigated the effects of siRNA electroporation on moDCs' viability, phenotype, migratory capacity, and ability to induce T-cell mediated immune responses. Finally, by using a standard duplex directed against the nuclear Lamins A and C we were able to demonstrate an efficient knockdown of a cellular messenger RNA in electroporated moDCs. We therefore propose siRNA electroporation into moDCs as an efficient method to manipulate DC function at large cell numbers without the use of chemical transfection reagents. This new approach represents an advantage especially in the light of clinical trials.

Cell Movement↗

PubMed2004

Efficient delivery of small interfering RNA for inhibition of IL-12p40 expression in vivo.

BACKGROUND: RNA interference is an evolutionary conserved immune response mechanism that can be used as a tool to provide novel insights into gene function and structure. The ability to efficiently deliver small interfering RNA to modulate gene expression in vivo may provide new therapeutic approaches to currently intractable diseases. METHODS: In vitro, siRNA targeting IL-12p40 was delivered to the murine macrophage cell line (J774A.1) encapsulated in a liposome with an IL-12 inducing agent (LPS/IFN-γ) over a number of time points. Controls included a variety of non-target specific siRNA reagents. Supernatants were analyzed for cytokine production while the cells were removed for mRNA profiling. In vivo, siRNA-targeting IL-12p40 was delivered to the murine peritoneal cavity in a therapeutic fashion, after endotoxin (LPS) challenge. Cells from the peritoneal cavity were removed by lavage and analyzed by flow cytometry. Levels of IL-12 present in lavage and in serum were also examined by ELISA. RESULTS: In this report, we show that IL-12p40 siRNA can specifically silence macrophage expression of IL-12p40 mRNA and IL-12p70 protein in vitro. We extend this finding to demonstrate that delivery of liposome encapsulated siRNA targeting IL-12p40 to the murine peritoneal cavity can modulate an inflammatory stimulus in vivo. Furthermore, specific siRNA can be used therapeutically after endotoxin challenge to reduce both the local and systemic inflammatory response. Thus, the delivery of siRNA can be used to elicit specific non-permanent inhibition of endogenous protein expression. CONCLUSION: In vitro silencing of IL-12p40 using siRNA at selected doses leads to specific knockdown of IL-12p70 protein production without inducing type I interferons. Furthermore, siRNA targeting murine IL-12p40 can be used therapeutically to counter an inflammatory response in vivo.

Journal Article↗

PubMed2005

A small interfering RNA targeting coxsackievirus B3 protects permissive HeLa cells from viral challenge.

We examined the ability of small interfering RNAs (siRNAs) to disrupt infection by coxsackievirus B3 (CVB3). The incorporation of siRNAs dramatically decreased cell death in permissive HeLa cells in parallel with a reduction in viral replication. Three of four siRNAs had potent anti-CVB3 activity. The present study thus demonstrates that the antiviral effect is due to the downregulation of viral replication. In addition, an effective CVB3-specific siRNA had similar antiviral effects in other related enteroviruses possessing sequence homology in the targeted region. Because the CVB3-specific siRNA is effective against other enteroviruses, siRNAs have potential for a universal anti-enterovirus strategy.

Antiviral Agents↗

PubMed2005

Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery for siRNAs toward treatment of bone-metastatic cancer. Accordingly, we report here that i.v. injection of GL3 luciferase siRNA complexed with atelocollagen showed effective reduction of luciferase expression from bone-metastatic prostate tumor cells developed in mouse thorax, jaws, and/or legs. We also show that the siRNA/atelocollagen complex can be efficiently delivered to tumors 24 h after injection and can exist intact at least for 3 days. Furthermore, atelocollagen-mediated systemic administration of siRNAs such as enhancer of zeste homolog 2 and phosphoinositide 3'-hydroxykinase p110-alpha-subunit, which were selected as candidate targets for inhibition of bone metastasis, resulted in an efficient inhibition of metastatic tumor growth in bone tissues. In addition, upregulation of serum IL-12 and IFN-alpha levels was not associated with the in vivo administration of the siRNA/atelocollagen complex. Thus, for treatment of bone metastasis of prostate cancer, an atelocollagen-mediated systemic delivery method could be a reliable and safe approach to the achievement of maximal function of siRNA.

Animals↗

PubMed2006

Suppression of bovine viral diarrhea virus replication by small interfering RNA and short hairpin RNA-mediated RNA interference.

Bovine viral diarrhea virus (BVDV) is a ubiquitous viral pathogen that affects cattle herds' worldwide causing significant economic loss. The current strategies to control BVDV infection include vaccination (modified-live or killed) and control of virus spread by enhanced biosecurity management, however, the disease remains prevalent. With the discovery of the sequence-specific method of gene silencing known as RNA interference (RNAi), a new era in antiviral therapies has begun. Here we report the efficient inhibition of BVDV replication by small interfering (siRNA) and short hairpin RNA (shRNA)-mediated gene silencing. siRNAs were generated to target the 5' non-translated (NTR) region and the regions encoding the C, NS4B and NS5A proteins of the BVDV genome. The siRNAs were first validated using an EGFP/BVDV reporter system and were then shown to suppress BVDV-induced cytopathic effects and viral titers in cell culture with surprisingly different activities compared to the reporter system. Efficient viral suppression was then achieved by bovine 7SK-expressed BVDV-specific shRNAs. Overall, our results demonstrated the use of siRNA and shRNA-mediated gene silencing to achieve efficient inhibition of the replication of this virus in cell culture.

Animals↗

PubMed2006

Hepatocyte nuclear factor-4alpha and -1 small interfering RNA inhibits hepatocyte differentiation induced by extracellular matrix.

Liver-specific genes and hepatocyte nuclear factor (HNF)-4alpha and -1 are coordinately regulated by extracellular matrix (ECM). However, still are unclear interactions between liver-specific genes and these liver-enriched transcription factors in the mechanism of hepatocyte differentiation regulated by ECM. To elucidate the relationship, we used small interfering RNA (siRNA), which obtains strong and specific knockdown of gene expression in cell culture. Treatment with siHNF-4alpha and siHNF-1 declined the expression levels for HNF-4alpha mRNA and HNF-1 mRNA in primary rat hepatocytes, respectively. The mRNA expressions of albumin, transthyretin, and apolipoproteins that were up-regulated in hepatocytes cultured on a basement membrane matrix, Engelbreth-Holm-Swarm (EHS) gel, were decreased in the presence of siHNF-4alpha or siHNF-1. Moreover, siHNF-4alpha and siHNF-1 did not affect the morphology and actin assembly of hepatocytes. These findings demonstrated that HNF-4alpha and HNF-1 directly regulate liver-specific gene expression and might be downstream of cytoskeletal organization in the mechanism by which the differentiated phenotype of hepatocytes is regulated by EHS gel.

Journal Article↗

PubMed2005

Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics.

Base Sequence↗