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Results for “RNA interference (RNAi)”

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PubMed2005

Nonviral genetic modification mediates effective transgene expression and functional RNA interference in human mesenchymal stem cells.

BACKGROUND: Human mesenchymal stem cells (hMSC) are increasingly the focus of both basic and clinical research due to their ability to strike a balance between self-renewal and commitment to mesodermal differentiation. However, the promising therapeutic utility of hMSC in regenerative medical approaches requires detailed knowledge about their molecular characteristics. Therefore, genetic modification of hMSC provides a powerful tool to understand their complex molecular regulation mechanisms. METHODS: Here we describe a proof of concept approach of separate and combined gene transfer and gene silencing by nonviral DNA transfection of enhanced green fluorescent protein (EGFP) and EGFP-targeted small interfering RNAs (siRNAs) in hMSC. For optimization of nonviral DNA and siRNA transfer different liposomal-based transfection strategies were validated. RESULTS: The highest fraction of EGFP-expressing hMSC was obtained using Lipofectamine 2000 (50%) which also mediated the highest transfection rates of siRNAs into hMSC (>or=92%). Stably EGFP-expressing hMSC maintained their proliferation capacity paired with the ability to differentiate into different mesodermal lineages (bone, cartilage, and fat) without loss of transgene expression. Based on our nonviral nucleic acid delivery technique we showed efficient, functional, and long-term RNA interference (RNAi) in hMSC by gene specific knock-down of transiently and stably expressed EGFP (88-98%). CONCLUSIONS: This is the first demonstration of efficient nonviral transfer of both nucleic acids (DNA and siRNA) into hMSC, exhibiting the potential of targeted modification of hMSC. In particular, the combination of these techniques represents a powerful gene transfer/silencing strategy, thus facilitating detailed genetic approaches to study regulatory networks in stem cell differentiation processes.

Cell Culture Techniques↗
PubMed2006

Transcription impairment and cell migration defects in elongator-depleted cells: implication for familial dysautonomia.

Mutations in IKBKAP, encoding a subunit of Elongator, cause familial dysautonomia (FD), a severe neurodevelopmental disease with complex clinical characteristics. Elongator was previously linked not only with transcriptional elongation and histone acetylation but also with other cellular processes. Here, we used RNA interference (RNAi) and fibroblasts from FD patients to identify Elongator target genes and study the role of Elongator in transcription. Strikingly, whereas Elongator is recruited to both target and nontarget genes, only target genes display histone H3 hypoacetylation and progressively lower RNAPII density through the coding region in FD cells. Interestingly, several target genes encode proteins implicated in cell motility. Indeed, characterization of IKAP/hELP1 RNAi cells, FD fibroblasts, and neuronal cell-derived cells uncovered defects in this cellular function upon Elongator depletion. These results indicate that defects in Elongator function affect transcriptional elongation of several genes and that the ensuing cell motility deficiencies may underlie the neuropathology of FD patients.

Carrier Proteins↗
PubMed2006

Small interfering RNA-mediated silencing of cytochrome P450 3A4 gene.

RNA interference (RNAi) is a specific and powerful tool used to manipulate gene expression and study gene function. The cytochrome P450 3A4 (CYP3A4) can metabolize more than 50% of drugs. In the present study, we investigated whether vector-expressed small interfering RNAs (siRNAs) altered the CYP3A4 expression and function using the Chinese hamster cell line (V79) overexpressing CYP3A4 (CHL-3A4). Three different siRNA oligonucleotides (3A4I, 3A4II, and 3A4III) were designed and tested for their ability to interfere with CYP3A4 gene expression. Our study demonstrated that transient transfection of CHL-3A4 cells with the 3A4III siRNAs, but not 3A4I and II, significantly reduced CYP3A4 mRNA levels by 65% and protein expression levels by 75%. All these siRNAs did not affect the expression of CYP3A5 at both mRNA and protein levels in V79 cells overexpressing CYP3A5. Transfection of CHL-3A4 cells with 3A4III siRNAs significantly diminished the cytotoxicity of two CYP3A4 substrate drugs, cyclophosphamide and ifosfamide, in CHL-3A4 cells, with the IC50 increased from 55 to 210 microM to >1000 microM. Nifedipine at 5.78, 14.44, and 28.88 microM was significantly (P < 0.01) depleted by approximately 100, 40, and 22%, respectively, in S9 fractions from CHL-3A4 cells compared with parental CHL-pIC19h cells. In addition, transfection of the CHL-3A4 cells with vectors expressing the 3A4III siRNAs almost completely inhibited CYP3A4-mediated nifedipine metabolism. This study demonstrated, for the first time, the specific suppression of CYP3A4 expression and function using vector-based RNAi technique. The use of RNAi is a promising tool for the study of cytochrome P450 family function.

Animals↗
PubMed2006

The assembly and maintenance of heterochromatin initiated by transgene repeats are independent of the RNA interference pathway in mammalian cells.

A role for the RNA interference (RNAi) pathway in the establishment of heterochromatin is now well accepted for various organisms. Less is known about its relevance and precise role in mammalian cells. We previously showed that tandem insertion of a 1,000-copy inducible transgene into the genome of baby hamster kidney (BHK) cells initiated the formation of an extremely condensed chromatin locus. Here, we characterized the inactive transgenic locus as heterochromatin, since it was associated with heterochromatin protein 1 (HP1), histone H3 trimethylated at lysine 9, and cytosine methylation in CpG dinucleotides. Northern blot analysis did not detect any transgene-derived small RNAs. RNAi-mediated Dicer knockdown did not disrupt the heterochromatic transgenic locus or up-regulate transgene expression. Moreover, neither Dicer knockdown nor overexpression of transgene-directed small interfering RNAs altered the bidirectional transition of the transgenic locus between the heterochromatic and euchromatic states. Interestingly, tethering of HP1 to the transgenic locus effectively induced transgene silencing and chromatin condensation in a Dicer-independent manner, suggesting a role for HP1 in maintaining the heterochromatic locus. Our results suggest that the RNAi pathway is not required for the assembly and maintenance of noncentromeric heterochromatin initiated by tandem transgene repeats in mammalian cells.

5' Untranslated Regions↗
PubMed2006

Highly efficient and specific modulation of cardiac calcium homeostasis by adenovector-derived short hairpin RNA targeting phospholamban.

Impaired function of the phospholamban (PLB)-regulated sarcoplasmic reticulum Ca(2+) pump (SERCA2a) contributes to cardiac dysfunction in heart failure (HF). PLB downregulation may increase SERCA2a activity and improve cardiac function. Small interfering (si)RNAs mediate efficient gene silencing by RNA interference (RNAi). However, their use for in vivo gene therapy is limited by siRNA instability in plasma and tissues, and by low siRNA transfer rates into target cells. To address these problems, we developed an adenoviral vector (AdV) transcribing short hairpin (sh)RNAs against rat PLB and evaluated its potential to silence the PLB gene and to modulate SERCA2a-mediated Ca(2+) sequestration in primary neonatal rat cardiomyocytes (PNCMs). Over a period of 13 days, vector transduction resulted in stable > 99.9% ablation of PLB-mRNA at a multiplicity of infection of 100. PLB protein gradually decreased until day 7 (7+/-2% left), whereas SERCA, Na(+)/Ca(2+) exchanger (NCX1), calsequestrin and troponin I protein remained unchanged. PLB silencing was associated with a marked increase in ATP-dependent oxalate-supported Ca(2+) uptake at 0.34 microM of free Ca(2+), and rapid loss of responsiveness to protein kinase A-dependent stimulation of Ca(2+) uptake was maintained until day 7. In summary, these results indicate that AdV-derived PLB-shRNA mediates highly efficient, specific and stable PLB gene silencing and modulation of active Ca(2+) sequestration in PNCMs. The availability of the new vector now enables employment of RNAi for the treatment of HF in vivo.

Animals↗
PubMed2004

Critical role of PIP5KI{gamma}87 in InsP3-mediated Ca(2+) signaling.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is the obligatory precursor of inositol 1,4,5-trisphosphate (InsP(3) or IP(3)) and is therefore critical to intracellular Ca(2+) signaling. Using RNA interference (RNAi), we identified the short splice variant of type I phosphatidylinositol 4-phosphate 5-kinase gamma (PIP5KIgamma87) as the major contributor of the PIP(2) pool that supports G protein-coupled receptor (GPCR)-mediated IP(3) generation. PIP5KIgamma87 RNAi decreases the histamine-induced IP(3) response and Ca(2+) flux by 70%. Strikingly, RNAi of other PIP5KI isoforms has minimal effect, even though some of these isoforms account for a larger percent of total PIP(2) mass and have previously been implicated in receptor mediated endocytosis or focal adhesion formation. Therefore, PIP5KIgamma87's PIP(2) pool that supports GPCR-mediated Ca(2+) signaling is functionally compartmentalized from those generated by the other PIP5KIs.

Calcium Signaling↗
PubMed2006

Essential role for Rac in heregulin beta1 mitogenic signaling: a mechanism that involves epidermal growth factor receptor and is independent of ErbB4.

Heregulins are a family of ligands for the ErbB3/ErbB4 receptors that play important roles in breast cancer cell proliferation and tumorigenesis. Limited information is available on the contribution of Rho GTPases to heregulin-mediated signaling. In breast cancer cells, heregulin beta1 (HRG) causes a strong activation of Rac; however, it does so with striking differences in kinetics compared to epidermal growth factor, which signals through ErbB1 (epidermal growth factor receptor [EGFR]). Using specific ErbB receptor inhibitors and depletion of receptors by RNA interference (RNAi), we established that, surprisingly, activation of Rac by HRG is mediated not only by ErbB3 and ErbB2 but also by transactivation of EGFR, and it is independent of ErbB4. Similar receptor requirements are observed for HRG-induced actin cytoskeleton reorganization and mitogenic activity via extracellular signal-regulated kinase (ERK). HRG-induced Rac activation was phosphatidylinositol 3-kinase dependent and Src independent. Furthermore, inactivation of Rac by expression of the Rac GTPase-activating protein beta2-chimerin inhibited HRG-induced ERK activation, mitogenicity, and migration in breast cancer cells. HRG mitogenic activity was also impaired by depletion of Rac1 using RNAi. Our studies established that Rac is a critical mediator of HRG mitogenic signaling in breast cancer cells and highlight additional levels of complexity for ErbB receptor coupling to downstream effectors that control aberrant proliferation and transformation.

Antibodies, Blocking↗
PubMed2006

Combinatorial RNA interference in Caenorhabditis elegans reveals that redundancy between gene duplicates can be maintained for more than 80 million years of evolution.

BACKGROUND: Systematic analyses of loss-of-function phenotypes have been carried out for most genes in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster. Although such studies vastly expand our knowledge of single gene function, they do not address redundancy in genetic networks. Developing tools for the systematic mapping of genetic interactions is thus a key step in exploring the relationship between genotype and phenotype. RESULTS: We established conditions for RNA interference (RNAi) in C. elegans to target multiple genes simultaneously in a high-throughput setting. Using this approach, we can detect the great majority of previously known synthetic genetic interactions. We used this assay to examine the redundancy of duplicated genes in the genome of C. elegans that correspond to single orthologs in S. cerevisiae or D. melanogaster and identified 16 pairs of duplicated genes that have redundant functions. Remarkably, 14 of these redundant gene pairs were duplicated before the divergence of C. elegans and C. briggsae 80-110 million years ago, suggesting that there has been selective pressure to maintain the overlap in function between some gene duplicates. CONCLUSION: We established a high throughput method for examining genetic interactions using combinatorial RNAi in C. elegans. Using this technique, we demonstrated that many duplicated genes can retain redundant functions for more than 80 million years of evolution. This provides strong support for evolutionary models that predict that genetic redundancy between duplicated genes can be actively maintained by natural selection and is not just a transient side effect of recent gene duplication events.

Animals↗
PubMed2006

Knock-down of hepatitis B virus X protein reduces the tumorigenicity of hepatocellular carcinoma cells.

Hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) in Southeast Asia and Hong Kong. Among the four proteins that are encoded by the HBV genome, HBV X (HBx) is the most potentially oncogenic factor. It is known that HBx plays an important role in hepatocarcinogenesis, but the exact functions and molecular mechanisms of HBx in HCC are not well understood. In this study, we constructed expression vectors for small hairpin RNAs (shRNA) against HBx and investigated their regulatory effects in PLC/PRF/5 HCC cells, which constitutively produce HBx. Our data show that this tool of RNA interference (RNAi) could successfully reduce the HBx mRNA and protein levels by 50-95%. RNAi targeting HBx in PLC/PRF/5 cells demonstrated significant reduction in cell proliferation, cell growth, anchorage-independent growth in soft agar, and tumour development in nude mice. In addition, depletion of HBx expression increased cell sensitivity to TNFalpha-mediated and serum-free-induced apoptosis, and reduced the expression levels of C-myc and Bcl-X(L). These findings suggest that HBx plays an important role in tumorigenicity and anti-apoptotic mechanisms in HCC.

Animals↗
PubMed2006

Analysis of gene expression profile in colon cancer using the Cancer Genome Anatomy Project and RNA interference.

OBJECTIVE: To investigate the changes in the gene expression profile in colon cancer to further identify gene markers that may be useful in the management of this disease. METHODS: Data from serial analysis of gene expression (SAGE) collected by the Cancer Genome Anatomy Project (CGAP) were used to detect the difference in gene expression between normal tissue and colon cancer, and were further confirmed in a sample of 20 patients using RT-PCR. To identify the functions of differential genes in regulating the cell growth of colon cancer, RNA interference (RNAi) was used to block one of these genes in the colon cancer cell line HCT-116. RESULTS: Expression changes of greater than twofold in two SAGE libraries of colon cancer compared to two of normal tissue were observed for 216 tags of a total of 195,160 transcript tags (54 up-regulated genes and 136 down-regulated genes). Subsequent analysis of 17 genes by RT-PCR confirmed the reliability of this analysis. RNAi-mediated blockage of one of these genes, transforming growth factor (TGF)beta1, significantly reduced the growth of a colon cancer cell line. CONCLUSIONS: The combination of CGAP analysis and RNAi provides an excellent system to rapidly define the specific genes that are up-regulated in cancer to impact the growth of cancer cells. Further study on these differential overexpressed genes may provide gene markers for the detection and treatment of colon cancer.

Biomarkers, Tumor↗
PubMed2005

Treating hepatitis C: can you teach old dogs new tricks?

Viruses depend on host-derived factors for their efficient genome replication. Here, we demonstrate that a cellular peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin B (CyPB), is critical for the efficient replication of the hepatitis C virus genome. CyPB interacted with the HCV RNA polymerase NS5B to directly stimulate its RNA binding activity. Both the RNA interference (RNAi)-mediated reduction of endogenous CyPB expression and the induced loss of NS5B binding to CyPB decreased the levels of HCV replication. Thus, CyPB functions as a stimulatory regulator of NS5B in HCV replication machinery. This regulation mechanism for viral replication identifies CyPB as a target for antiviral therapeutic strategies.

Antiviral Agents↗
PubMed2006

p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation.

It has been reported that genomic DNA methylation decreases gradually during cell culture and an organism's aging. However, less is known about the methylation changes of age-related specific genes in aging. p21(Waf1/Cip1) and p16(INK4a) are cyclin-dependent kinase (Cdk) inhibitors that are critical for the replicative senescence of normal cells. In this study, we show that p21(Waf1/Cip1) and p16(INK4a) have different methylation patterns during the aging process of normal human 2BS and WI-38 fibroblasts. p21(Waf1/Cip1) promoter is gradually methylated up into middle-aged fibroblasts but not with senescent fibroblasts, whereas p16(INK4a) is always unmethylated in the aging process. Correspondently, the protein levels of DNA methyltransferase 1 (DNMT1) and DNMT3a increase from young to middle-aged fibroblasts but decrease in the senescent fibroblasts, while DNMT3b decreases stably from young to senescent fibroblasts. p21(Waf1/Cip1) promoter methylation directly represses its expression and blocks the radiation-induced DNA damage-signaling pathway by p53 in middle-aged fibroblasts. More importantly, demethylation by 5-aza-CdR or DNMT1 RNA interference (RNAi) resulted in an increased p21(Waf1/Cip1) level and premature senescence of middle-aged fibroblasts demonstrated by cell growth arrest and high beta-Galactosidase expression. Our results suggest that p21(Waf1/Cip1) but not p16(INK4a) is involved in the DNA methylation mediated aging process. p21(Waf1/Cip1) promoter methylation may be a critical biological barrier to postpone the aging process.

Azacitidine↗
PubMed2006

Progress in the development of nucleic acid therapeutics.

Abnormal gene expression is a hallmark of many diseases. Gene-specific downregulation of aberrant genes could be useful therapeutically and potentially less toxic than conventional therapies due its specificity. Over the years, many strategies have been proposed for silencing gene expression in a gene-specific manner. Three major approaches are antisense oligonucleotides (AS-ONs), ribozymes/DNAzymes, and RNA interference (RNAi). In this brief review, we will discuss the successes and shortcomings of these three gene-silencing methods, and the approaches being taken to improve the effectiveness of antisense molecules. We will also provide an overview of some of the clinical applications of antisense therapy.

Animals↗
PubMed2006

Inhibition of CD147 gene expression via RNA interference reduces tumor cell invasion, tumorigenicity and increases chemosensitivity to paclitaxel in HO-8910pm cells.

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN or CD147), a member of the immunoglobulin family and a glycoprotein enriched on the surface of tumor cells, promotes invasion, metastasis, growth and survival of malignant cells, and confers resistance to some chemotherapeutic drugs. Here, we used a human U6 promoter-driven DNA template approach to induce short hairpin RNA (shRNA)-triggered RNA interference (RNAi) to block CD147 gene expression in the human ovarian cancer cell line HO-8910pm. Knockdown of CD147 by shRNA resulted in decrease of the HO-8910pm invasion activity in vitro and tumorigenicity in nude mice. The suppression of CD147 expression also sensitized cells to be more sensitive to paclitaxel. These results suggested that CD147 was an ovarian cancer-related gene and CD147 might be a potential target for therapeutic anti-cancer drugs.

Animals↗
PubMed2004

Lumen morphogenesis in C. elegans requires the membrane-cytoskeleton linker erm-1.

Epithelial tubes are basic building blocks of complex organs, but their architectural requirements are not well understood. Here we show that erm-1 is a unique C. elegans ortholog of the ERM family of cytoskeleton-membrane linkers, with an essential role in lumen morphogenesis. ERM-1 localizes to the luminal membranes of those tubular organ epithelia which lack stabilization by cuticle. RNA interference (RNAi), a germline deletion, and overexpression of erm-1 cause cystic luminal phenotypes in these epithelia. Confocal and ultrastructural analyses indicate that erm-1 functions directly in apical membrane morphogenesis, rather than in epithelial polarity and junction assembly as has been previously proposed for ERMs. We also show that act-5/cytoplasmic actin and sma-1/beta-H-spectrin are required for lumen formation and functionally interact with erm-1. Our findings suggest that there are common structural constraints on the architecture of diverse organ lumina.

Animals↗
PubMed2004

'Knock down' of DNA polymerase beta by RNA interference: recapitulation of null phenotype.

DNA polymerase beta (pol beta) is the major DNA polymerase involved in the base excision repair (BER) pathway in mammalian cells and, as a consequence, BER is severely compromised in cells lacking pol beta. Pol beta null (-/-) mouse embryos are not viable and pol beta null cells are hypersensitive to alkylating agents. Using RNA interference (RNAi) technology in mouse cells, we have reduced the pol beta protein and mRNA to undetectable levels. Pol beta knockdown cell lines display a pattern of hypersensitivity to DNA damaging agents similar to that observed in pol beta null cells. Generation of pol beta knock down cells makes it possible to combine the pol beta null phenotype with deficiencies in other DNA repair proteins, thereby helping to elucidate the role of pol beta and its interactions with other proteins in mammalian cells.

Animals↗
PubMed2004

The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin alpha.

Addition of N-acetylglucosamine (GlcNAc) is a ubiquitous form of intracellular glycosylation catalyzed by the conserved O-linked GlcNAc transferase (OGT). OGT contains an N-terminal domain of tetratricopeptide (TPR) repeats that mediates the recognition of a broad range of target proteins. Components of the nuclear pore complex are major OGT targets, as OGT depletion by RNA interference (RNAi) results in the loss of GlcNAc modification at the nuclear envelope. To gain insight into the mechanism of target recognition, we solved the crystal structure of the homodimeric TPR domain of human OGT, which contains 11.5 TPR repeats. The repeats form an elongated superhelix. The concave surface of the superhelix is lined by absolutely conserved asparagines, in a manner reminiscent of the peptide-binding site of importin alpha. Based on this structural similarity, we propose that OGT uses an analogous molecular mechanism to recognize its targets.

Acetylglucosamine↗
PubMed2006

RNA interference-mediated gene silencing of pleiotrophin through polyethylenimine-complexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts.

RNA interference (RNAi) is a powerful strategy to inhibit gene expression through specific mRNA degradation mediated by small interfering RNAs (siRNAs). In vivo, however, the application of siRNAs is severely limited by their instability and poor delivery into target cells and target tissues. Glioblastomas are the most frequent and malignant brain tumors with, so far, limited treatment options. To develop novel and more efficacious therapies, advanced targeting strategies against glioblastoma multiforme (GBM)-relevant target genes must be established in vivo. Here we use RNAi-based targeting of the secreted growth factor pleiotrophin (PTN), employing a polyethylenimine (PEI)/siRNA complex strategy. We show that the complexation of chemically unmodified siRNAs with PEI leads to the formation of complexes that condense and completely cover siRNAs as determined by atomic force microscopy (AFM). On the efficient cellular delivery of these PEI/siRNA complexes, the PTN downregulation in U87 glioblastoma cells in vitro results in decreased proliferation and soft agar colony formation. More importantly, in vivo treatment of nude mice through systemic application (subcutaneous or intraperitoneal) of PEI-complexed PTN siRNAs leads to the delivery of intact siRNAs into subcutaneous tumor xenografts and a significant inhibition of tumor growth without a measurable induction of siRNA-mediated immunostimulation. Likewise, in a clinically more relevant orthotopic mouse glioblastoma model with U87 cells growing intracranially, the injection of PEI-complexed PTN siRNAs into the CNS exerts antitumoral effects. In conclusion, we present the PEI complexation of siRNAs as a universally applicable platform for RNAi in vitro and in vivo and establish, also in a complex and relevant orthotopic tumor model, the potential of PEI/siRNA-mediated PTN gene targeting as a novel therapeutic option in GBM.

Animals↗