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Results for “RNAi pathway”

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At least 343 records · Page 19Linked to original sources
PubMed2005

RNA interference (RNAi) for extracellular signal-regulated kinase 1 (ERK1) alone is sufficient to suppress cell viability in ovarian cancer cells.

While ovarian cancer is a leading cause of death in females today, the molecular, genetic, and environmental factors that initiate and support the progression of this disease are still only partially understood. The extracellular signal-regulated kinase (ERK) signaling pathway is a major contributor to cellular growth, differentiation and survival. Recently, we reported that this pathway is constitutively activated in ovarian cancer cells, and that by using RNA interference (RNAi) for ERK1 and ERK2, we were able to significantly suppress the number of viable tumor cells. In the present study, we have further investigated the mechanisms by which RNAi for the ERK kinases decreased viability in these cancer cells. It was determined that treatment of the cancer cells with small inhibitory RNAs (siRNAs) directed against ERK1 and ERK2 leads to the induction of apoptosis and necrosis by four hours following treatment. Additionally, we found that primary, nonmalignant ovarian cells do not respond similarly to ERK siRNA treatment and that these cells fail to die following treatment. Data presented show that ERK2 expression is more difficult to silence, depending upon cell type being examined and that silencing ERK1 expression alone is sufficient to significantly decrease tumor cell viability.

Apoptosis↗
PubMed2005

Suppressor of Hairless and Presenilin phenotypes imply involvement of canonical Notch-signalling in segmentation of the spider Cupiennius salei.

Arthropods, vertebrates, and annelids all have a segmented body. Our recent discovery of involvement of Notch-signalling in spider segmentation revived the discussion on the origin of segmented body plans and suggests the sharing of a common genetic program in a common ancestor. Here, we analysed the spider homologues of the Suppressor of Hairless and Presenilin genes, which encode components of the canonical Notch-pathway, to further explore the role of Notch-signalling in spider segmentation. RNAi silencing of two spider Suppressor of Hairless homologues and the spider Presenilin homologue causes severe segmentation phenotypes. The most prominent defect is the consistent breakdown of segmentation after the formation of three (Suppressor of Hairless) or five (Presenilin) opisthosomal segments. These phenotypes indicate that Notch-signalling during spider segmentation likely involves the canonical pathway via Presenilin and Suppressor of Hairless. Furthermore, it implies that Notch-signalling influences both the formation and patterning of the spider segments: it is required for the specification of the posterior segments and for proper specification of the segment boundaries. We argue that alternative, partly redundant, pathways might act in the formation of the anterior segments that are not active in the posterior segments. This suggests that at least some differences exist in the specification of anterior and posterior segments of the spider, a finding that may be valid for most short germ arthropods. Our data provide additional evidence for the similarities of Notch-signalling in spider segmentation and vertebrate somitogenesis and strengthen our previous notion that the formation of the segments in arthropods and vertebrates might have shared a genetic program in a common ancestor.

Amino Acid Sequence↗
PubMed2005

Cancer-specific functions of SIRT1 enable human epithelial cancer cell growth and survival.

SIRT1 is a conserved NAD-dependent deacetylase that regulates life span in accord with nutritional provision. In mammalian cells, SIRT1 also down-regulates stress-induced p53 and FoxO pathways for apoptosis, thus favoring survival under stress. The functioning of SIRT1 under normal, nonstressed conditions of cell growth is unknown. Here we have asked if SIRT1 has the capacity to influence cell viability in the absence of applied stress. For this purpose we used synthetic small interfering RNA to silence SIRT1 gene expression by RNA interference (RNAi). We show that the process of RNAi, by itself, does not affect cell growth and is not sufficient to activate a cellular stress response (indicated by lack of activation of endogenous p53). We also show that, in the absence of applied stress, SIRT1 silencing induces growth arrest and/or apoptosis in human epithelial cancer cells. In contrast, normal human epithelial cells and normal human diploid fibroblasts seem to be refractory to SIRT1 silencing. Combined gene knockout with RNAi cosilencing experiments indicate that SIRT1 and Bcl-2 may suppress separable apoptotic pathways in the same cell lineage and that the SIRT1-regulated pathway is independent of p53, Bax, and caspase-2. Alternatively, SIRT1 may suppress apoptosis downstream from these apoptotic factors. In either case, we show that FoxO4 (but not FoxO3) is required as proapoptotic mediator. We further identify caspase-3 and caspase-7 as downstream executioners of SIRT1/FoxO4-regulated apoptosis. Our work identifies SIRT1 as a novel target for selective killing of cancer versus noncancer epithelial cells.

Apoptosis↗
PubMed2003

The Drosophila IgC2 domain protein Friend-of-Echinoid, a paralogue of Echinoid, limits the number of sensory organ precursors in the wing disc and interacts with the Notch signaling pathway.

The Notch signaling pathway is critical in cell fate specification throughout development. In the developing wing disc, single sensory organ precursors (SOPs) are selected from proneural clusters via a process of lateral inhibition mediated by the Notch signaling pathway. The epidermal growth factor receptor (EGFR) pathway has also been implicated in SOP formation. Here, we describe the Drosophila melanogaster gene friend of echinoid (fred), a paralogue of echinoid (ed), a gene recently identified as a negative regulator of the EGFR pathway. fred function was examined in transgenic flies by using inducible RNA interference (RNAi). Suppression of fred in developing wing discs results in specification of ectopic SOPs, additional microchaeta, and cell death. In eye-antennal discs, fred suppression causes a rough eye phenotype. These phenotypes are suppressed by overexpression of Notch, Suppressor of Hairless [Su(H)], and Enhancer of split m7. In contrast, overexpression of Hairless, a negative regulator of the Notch pathway, and decreased Su(H) activity enhance these phenotypes. Thus, fred acts in close concert with the Notch signaling pathway. Dosage-sensitive genetic interaction also suggests a close relationship between fred and ed.

Amino Acid Sequence↗
PubMed2005

Application of RNAi to cancer research and therapy.

The introduction of double-stranded RNA (dsRNA) into cells can effectively and specifically lead to the degradation of corresponding mRNAs in a gene-dependent manner, which is defined as RNA interfering (RNAi). This powerful technology has been widely employed to manipulate gene expression in mammalian and human cells, elucidate signal pathways and identify gene functions in a whole-genome scale. Simultaneously, many pharmaceutical companies are very interested in the research and development of RNAi-based drugs for various diseases, especially in cancers. In present review, we attempt to recapitulate the potential application of this breakthrough technology in many aspects of cancer gene therapy such as genome-scale screens, target identification and validation, functional analysis and animal models for diverse diseases.

Animals↗
PubMed2002

ARGONAUTE1 is required for efficient RNA interference in Drosophila embryos.

Double-stranded RNA (dsRNA) triggers homology-dependent posttranscriptional gene interference (RNAi) in a diverse range of eukaryotic organisms, in a process mechanistically related to viral and transgene-mediated cosuppression. RNAi is characterized by the conversion of long dsRNA into approximately 21-25-nt small interfering RNAs (siRNA) that guide the degradation of homologous mRNA. Many of the genes required for siRNA production and target mRNA degradation are widely conserved. Notably, members of the Argonaute-like gene family from Arabidopsis, Caenorhabditis elegans, Drosophila, and Neurospora have been genetically and/or biochemically identified as components of the RNAi/cosuppression pathway. We show here that mutations in the Drosophila Argonaute1 (AGO1) gene suppress RNAi in embryos. This defect corresponds to a reduced ability to degrade mRNA in response to dsRNA in vitro. Furthermore, AGO1 is not required for siRNA production in vitro nor can the introduction of siRNA bypass AGO1 mutants in vivo. These data suggest that AGO1 functions downstream of siRNA production.

ATP-Binding Cassette Transporters↗
PubMed2002

RNAi functions in cultured mammalian neurons.

In a wide range of organisms, double-stranded RNA triggers posttranscriptional gene silencing or RNA interference (RNAi). Small interfering RNAs, the 21-nt double-stranded RNA intermediates of this natural pathway, have became a powerful tool to knock down specific gene expression in mammalian cell lines and potentially will be useful for the analysis of loss-of-function phenotypes. In mammalian primary neuronal cultures, where genetic manipulations are especially difficult, RNAi might be developed into a highly efficacious tool to study the roles of specific genes in neuron development and functioning. Neurons, however, have been considered the most resistant to RNAi. We report here an application of RNAi to postmitotic primary neuronal cultures. Synthetic siRNA can be readily introduced into neurons and effectively inhibit the expression of endogenous and transfected genes.

Animals↗
PubMed2005

Csk homologous kinase (CHK), unlike Csk, enhances MAPK activation via Ras-mediated signaling in a Src-independent manner.

Substantial evidence exists supporting the notion that Csk and CHK, two negative regulatory kinases of the Src tyrosine kinase family, play distinct roles during development of the nervous system. One of the differences relies on the effects of both kinases on the MAPK transduction pathway. Specifically, CHK was shown to enhance MAPK signaling, while the role of Csk was unclear. In this work, we compared the effect of CHK versus Csk on MAPK signaling and elucidated the signaling pathway mediated by CHK leading to the activation of Erk1/2. Exogenous expression of wild-type CHK, but not Csk or a dead-kinase mutant of CHK, resulted in enhanced Erk1/2 phosphorylation in PC12 cells. CHK inhibited Src activity following stimulation of the cells with NGF. However, stimulation of Erk1/2 activation by CHK was independent of the NGF stimulation or the inhibition of Src kinase by CHK. CHK induced a complex formation between SHP-2 and Grb2, subsequently leading to the increased activity of Ras as well as Erk1/2 activation via the Raf/MEK1/2 pathway. Down-regulation of the expression of endogenous CHK by RNAi in PC12 cells led to a significant decrease in MAPK activation following NGF stimulation. Stimulation of CHK-overexpressing PC12 cells with EGF induced neurite outgrowth in the majority of cells. Taken together, this study describes for the first time the Src-independent actions of CHK and provides novel insights into CHK function in neural cells.

Adenoviridae↗
PubMed2006

A genome-wide Drosophila RNAi screen identifies DYRK-family kinases as regulators of NFAT.

Precise regulation of the NFAT (nuclear factor of activated T cells) family of transcription factors (NFAT1-4) is essential for vertebrate development and function. In resting cells, NFAT proteins are heavily phosphorylated and reside in the cytoplasm; in cells exposed to stimuli that raise intracellular free Ca2+ levels, they are dephosphorylated by the calmodulin-dependent phosphatase calcineurin and translocate to the nucleus. NFAT dephosphorylation by calcineurin is countered by distinct NFAT kinases, among them casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Here we have used a genome-wide RNA interference (RNAi) screen in Drosophila to identify additional regulators of the signalling pathway leading from Ca2+-calcineurin to NFAT. This screen was successful because the pathways regulating NFAT subcellular localization (Ca2+ influx, Ca2+-calmodulin-calcineurin signalling and NFAT kinases) are conserved across species, even though Ca2+-regulated NFAT proteins are not themselves represented in invertebrates. Using the screen, we have identified DYRKs (dual-specificity tyrosine-phosphorylation regulated kinases) as novel regulators of NFAT. DYRK1A and DYRK2 counter calcineurin-mediated dephosphorylation of NFAT1 by directly phosphorylating the conserved serine-proline repeat 3 (SP-3) motif of the NFAT regulatory domain, thus priming further phosphorylation of the SP-2 and serine-rich region 1 (SRR-1) motifs by GSK3 and CK1, respectively. Thus, genetic screening in Drosophila can be successfully applied to cross evolutionary boundaries and identify new regulators of a transcription factor that is expressed only in vertebrates.

Animals↗
PubMed2006

JNK- and Fos-regulated Mmp1 expression cooperates with Ras to induce invasive tumors in Drosophila.

Loss of the epithelial polarity gene scribble in clones of Drosophila imaginal disc cells can cooperate with Ras signaling to induce malignant tumors. Such mutant tissue overproliferates, resists apoptosis, leaves its place of origin and invades other organs, ultimately causing lethality. We show that increased Jun N-terminal kinase (JNK) signaling resulting from the loss of scribble promotes the movement of transformed cells to secondary sites. This effect requires Fos-dependent transcriptional activation of a matrix metalloprotease gene mmp1 downstream of JNK. Expression of the Mmp inhibitor Timp or Mmp RNAi knockdown suppresses cell invasiveness. The proinvasive function of the JNK pathway is revealed in a tumor context when active Ras signaling prevents the apoptotic response to JNK activity as it occurs in nontransformed cells. Based on these results, we present a model that explains the oncogenic cooperation between JNK and Ras, and describes how aberrant regulation of cell survival, proliferation and mobilization cooperate to incite malignant tumor formation.

Animals↗
PubMed2005

Functional genomic analysis of RNA interference in C. elegans.

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.

Amino Acid Motifs↗
PubMed2003

The Ref/Aly proteins are dispensable for mRNA export and development in Caenorhabditis elegans.

The mRNA export pathway is highly conserved throughout evolution. We have used RNA interference (RNAi) to functionally characterize bona fide RNA export factors and components of the exon-exon junction complex (EJC) in Caenorhabditis elegans. RNAi of CeNXT1/p15, the binding partner of CeNXF1/TAP, caused early embryonic lethality, demonstrating an essential function of this gene during C. elegans development. Moreover, depletion of this protein resulted in nuclear accumulation of poly(A)(+) RNAs, supporting a direct role of NXT1/p15 in mRNA export in C. elegans. Previously, we have shown that RNAi of CeSRm160, a protein of the EJC complex, resulted in wild-type phenotype; in the present study, we demonstrate that RNAi of CeY14, another component of this complex, results in embryonic lethality. In contrast, depletion of the EJC component CeRNPS1 results in no discernible phenotype. Proteins of the REF/Aly family act as adaptor proteins mediating the recruitment of the mRNA export factor, NXF1/TAP, to mRNAs. The C. elegans genome encodes three members of the REF/Aly family. RNAi of individual Ref genes, or codepletion of two Ref genes in different combinations, resulted in wild-type phenotype. Simultaneous suppression of all three Ref genes did not compromise viability or progression through developmental stages in the affected progeny, and only caused a minor defect in larval mobility. Furthermore, no defects in mRNA export were observed upon simultaneous depletion of all three REF proteins. These results suggest the existence of multiple adaptor proteins that mediate mRNA export in C. elegans.

Amino Acid Sequence↗
PubMed2005

The deubiquitinating enzyme USP1 regulates the Fanconi anemia pathway.

Protein ubiquitination and deubiquitination are dynamic processes implicated in the regulation of numerous cellular pathways. Monoubiquitination of the Fanconi anemia (FA) protein FANCD2 appears to be critical in the repair of DNA damage because many of the proteins that are mutated in FA are required for FANCD2 ubiquitination. By screening a gene family RNAi library, we identify the deubiquitinating enzyme USP1 as a novel component of the Fanconi anemia pathway. Inhibition of USP1 leads to hyperaccumulation of monoubiquitinated FANCD2. Furthermore, USP1 physically associates with FANCD2, and the proteins colocalize in chromatin after DNA damage. Finally, analysis of crosslinker-induced chromosomal aberrations in USP1 knockdown cells suggests a role in DNA repair. We propose that USP1 deubiquitinates FANCD2 when cells exit S phase or recommence cycling after a DNA damage insult and may play a critical role in the FA pathway by recycling FANCD2.

Animals↗
PubMed2026

Integrated RNA-seq and RNAi analyses reveal that ABCF2 is involved in defense against Vibrio parahaemolyticus in Penaeus vannamei.

The sustainable development of shrimp aquaculture is significantly compromised by Vibrio parahaemolyticus infections. Identifying host resistance genes and characterizing their immunological roles are essential for developing effective disease control strategies. In this study, we conducted a comparative transcriptomic analysis of intestinal tissues from Penaeus vannamei exhibiting varying degrees of pathological damage post-V. parahaemolyticus challenge to identify key resistance genes. KEGG enrichment analysis revealed that the ABC transporter pathway was markedly enriched among upregulated genes in both the 9 h vs 0 h and 48 h vs 0 h comparison groups. Based on the expression profiles and domain characteristics of genes within this pathway, the full transporter PvABCA3, half transporter PvABCC1, and soluble protein PvABCF2 were selected for RNAi assays. The result indicated that silencing PvABCF2, but not PvABCA3 and PvABCC1, significantly increased mortality, tissue damage, and Vibrio load in V. parahaemolyticus-challenged shrimp. Further investigation revealed that PvABCF2 silencing substantially suppressed the expression of antimicrobial peptides (AMPs), components of the proPO-activating system, and key genes involved in the JAK-STAT and NF-κB signaling pathways. These findings suggested that the increased susceptibility of shrimp to V. parahaemolyticus following PvABCF2 silencing may be associated with downregulation of these specific immune-related genes. Moreover, one SNP within PvABCF2 was found to be markedly associated with resistance to V. parahaemolyticus via SNP association analysis. Collectively, these results suggested that PvABCF2 was involved in defense response against V. parahaemolyticus and identified a potential molecular marker for disease-resistant breeding.

Animals↗
PubMed2004

RNA interference: a potent tool for gene-specific therapeutics.

RNA interference (RNAi) is a process through which double-stranded RNA induces the activation of cellular pathways, leading to potent and selective silencing of genes with homology to the double strand. Much excitement surrounding small interfering RNA (siRNA)-mediated therapeutics arises from the fact that this approach overcomes many of the shortcomings previously experienced with approaches such as antibodies, antisense oligonucleotides and pharmacological inhibitors. Induction of RNAi through administration of siRNA has been successfully used in treatment of hepatitis, viral infections, and cancer. In this review we will present a brief history of RNAi, methods of inducing RNAi, application of RNAi in the therapeutic setting, and the possibilities of using this highly promising approach in the context of transplantation.

Animals↗
PubMed2005

Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila.

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, we carried out a genome-wide RNA interference (RNAi) screen in cultured Drosophila cells. We identified 121 genes whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, we found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, we identified a key negative regulator of Drosophila JAK/STAT signaling, protein tyrosine phosphatase PTP61F, and showed that it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. Our study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway.

Active Transport, Cell Nucleus↗
PubMed2006

[Research advances on the relationship of PI3-kinase/Akt/mTOR pathway and epigenetic modification].

The mammalian target of rapamycin (mTOR) has been shown to link growth factor signaling and posttranscriptional control of protein translation through activation of the PI3K/Akt pathway, which is frequently involved in cell cycle progression. The inhibition of mTOR has promising potential in anticancer and immunosuppressive therapies, and additional phase II clinical trials are ongoing. Epigenetic modification, which involves DNA methylation, histone modification and chromatin remodeling, as well as the recently described RNAi mechanism, can initiate the formation of silenced chromatin. Persistent activation or inhibition of the mTOR pathway may affect epigenetic modification. In this paper, we reviewed the research advances in the relationship between PI3K/Akt/mTOR and epigenetic modification.

Animals↗
PubMed2006

PHTS, a novel putative tumor suppressor, is involved in the transformation reversion of HeLaHF cells independently of the p53 pathway.

HeLaHF is a non-transformed revertant of HeLa cells, likely resulting from the activation of a putative tumor suppressor(s). p53 protein was stabilized in this revertant and reactivated for certain transactivation functions. Although p53 stabilization has not conclusively been linked to the reversion, it is clear that the genes in p53 pathway are involved. The present study confirms the direct role of p53 in HeLaHF reversion by demonstrating that RNAi-mediated p53 silencing partially restores anchorage-independent growth potential of the revertant through the suppression of anoikis. In addition, we identified a novel gene, named PHTS, with putative tumor suppressor properties, and showed that this gene is also involved in HeLaHF reversion independently of the p53 pathway. Expression profiling revealed that PHTS is one of the genes that is up-regulated in HeLaHF but not in HeLa. It encodes a putative protein with CD59-like domains. RNAi-mediated PHTS silencing resulted in the partial restoration of transformation (anchorage-independent growth) in HeLaHF cells, similar to that of p53 gene silencing, implying its tumor suppressor effect. However, the observed increased transformation potential by PHTS silencing appears to be due to an increased anchorage-independent proliferation rate rather than suppression of anoikis, unlike the effect of p53 silencing. p53 silencing did not affect PHTS gene expression, and vice versa, suggesting PHTS may function in a new and p53-independent tumor suppressor pathway. Furthermore, over-expression of PHTS in different cancer cell lines, in addition to HeLa, reduces cell growth likely via induced apoptosis, confirming the broad PHTS tumor suppressor properties.

Amino Acid Sequence↗