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PubMed2004

[Experimental research of targeting hTERT gene inhibited in hepatocellular carcinoma therapy by RNA interference].

BACKGROUND & OBJECTIVE: RNA interference (RNAi) is a new gene blocking technology that silences target gene at post-transcription level induced by the small interference RNA (siRNA). RNAi has been demonstrated great prospect in gene functional research and gene therapy areas. Nowadays, RNAi has been reported to be used to inhibit the expression of endogenous genes including cyclophilin, GAPDH, p53, and c-myc; and there were some progresses in the therapy of the diseases caused by AIDS and hepatitis viruses with RNAi. However, hTERT gene, which was highly expressed in hepatocellular carcinoma and other malignant neoplasm, has not been researched by RNAi. In present research, we utilized RNAi to inhibit hTERT gene expression in vitro and in vivo, investigated the feasibility and specificity of gene therapy for hepatocellular carcinoma. METHODS: Small interference RNAs homologous to hTERT gene were designed,pTZU6+1-shRNA-hTERT vector was constructed and transfected into hepatocellular carcinoma SMMC-7721 cells and transplanted SMMC-7721 tumor in nude mice to induce RNAi. The changes of hTERT gene expression and tumor cell proliferation in both siRNA treatment groups and control group were determined by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), immunochemistry in vitro and in vivo. RESULTS: The expression of hTERT had been obviously inhibited by RNAi in vitro. The inhibition rate of cell growth was 37.5% after pTZU6+1-shRNA-hTERT vector was transfected to hepatocellular carcinoma SMMC-7721 cells; the phase of cell cycle indicated the reduction of S phase, while G(1)/G(0) phase increased. The mRNA level of hTERT decreased from 99.4% to 53.1%, its protein expression reduced from 86.3% to 46.6%. The tumor size reduced after treated with pTZU6+1-shRNA-hTERT vector in vivo; hTERT mRNA level decreased from 99.1% to 76.2%, and its protein expression decreased from 87.2% to 61.8% in siRNA treatment group. In contrast, there were no changes in control groups in vitro and in vivo. CONCLUSION: RNAi inhibits the hTERT gene expression and proliferation of hepatocellular carcinoma SMMC-7721 cells with specificity, and is a possible new approach for neoplasm gene therapy.

Animals↗
PubMed2003

A micrococcal nuclease homologue in RNAi effector complexes.

RNA interference (RNAi) regulates gene expression by the cleavage of messenger RNA, by mRNA degradation and by preventing protein synthesis. These effects are mediated by a ribonucleoprotein complex known as RISC (RNA-induced silencing complex). We have previously identified four Drosophila components (short interfering RNAs, Argonaute 2 (ref. 2), VIG and FXR) of a RISC enzyme that degrades specific mRNAs in response to a double-stranded-RNA trigger. Here we show that Tudor-SN (tudor staphylococcal nuclease)--a protein containing five staphylococcal/micrococcal nuclease domains and a tudor domain--is a component of the RISC enzyme in Caenorhabditis elegans, Drosophila and mammals. Although Tudor-SN contains non-canonical active-site sequences, we show that purified Tudor-SN exhibits nuclease activity similar to that of other staphylococcal nucleases. Notably, both purified Tudor-SN and RISC are inhibited by a specific competitive inhibitor of micrococcal nuclease. Tudor-SN is the first RISC subunit to be identified that contains a recognizable nuclease domain, and could therefore contribute to the RNA degradation observed in RNAi.

Animals↗
PubMed2003

R2D2, a bridge between the initiation and effector steps of the Drosophila RNAi pathway.

The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). The siRNA-generating enzyme was purified from Drosophila S2cells and consists of two stoichiometric subunits: Dicer-2(DCR-2) and a previously unknown protein that we named R2D2. R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of DCR-2. Rather, the DCR-2/R2D2 complex, but not DCR-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer to RISC.

Amino Acid Sequence↗
PubMed2006

Knockdown of Sox4 expression by RNAi induces apoptosis in ACC3 cells.

Microarray RNA gene expression profiling analysis has shown that Sox4 (Sry-related high mobility group (HMG) box 4) is one of the most upregulated genes in adenoid cystic carcinoma (ACC), relative to non-neoplastic tissue of origin. Here, we show that Sox4 protein is similarly upregulated in ACC by immunohistochemistry of 28 primary cancers and 20 normal tissues. To elucidate the functional significance of these findings, RNA interference (RNAi)-mediated RNA silencing was used to downregulate Sox4 expression in the ACC-derived cell line, ACC3. With confirmed knockdown of Sox4 protein, cell viability was reduced by 51%, with a corresponding increase of apoptosis to 85% as compared to 12% in controls. Apoptosis was confirmed by cell morphology, DNA fragmentation and flow cytometry. Cells could be rescued from the proapoptotic effects of Sox4 RNAi by co-transfection with a construct expressing functional Sox4. Microarray gene expression profiling of RNAi knockdown experiments shows that downregulation of Sox4-modulated expression of critical genes involved in apoptosis and cell cycle control. Overall, our findings suggest that Sox4 contributes to the malignant phenotype of ACC cells by promoting cell survival.

Apoptosis↗
PubMed2004

Tandem inverted repeat system for selection of effective transgenic RNAi strains in Chlamydomonas.

RNA interference (RNAi), the double-stranded RNA (dsRNA) triggered post-transcriptional gene silencing, is becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of inverted repeat (IR) transgenes, has been achieved in protozoa, algae, fungi, plants, and metazoans. However, the level of gene silencing is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. This is a hindrance in functional genomics studies, where it is desirable to suppress target genes reliably to analyze unknown phenotypes. Consequently, we explored strategies for direct selection of effective transgenic RNAi lines in Chlamydomonas reinhardtii. We initially attempted to suppress expression of the Rubisco small subunit multigene family by placing an IR, homologous to the conserved coding sequence, in the 3'UTR of a transgene conferring resistance to bleomycin. However, this approach was fairly inefficient at inducing RNAi as many strains displayed defective transgene integration, resulting in partial or complete deletion of the IR, or low levels of dsRNA expression, presumably due to transcriptional silencing of the integrated IR transgenes. To overcome these problems we designed a system consisting of tandem IR transgenes that consistently triggered co-silencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase beta subunit) and another gene of interest (encoding either Ku80, an RNA-binding protein, or a thioredoxin isoform). We anticipate that this approach will be useful for generating stable hypomorphic epi-mutants in high-throughput phenotypic screens.

Animals↗
PubMed2003

Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila.

RNA interference (RNAi) designates the multistep process by which double-stranded RNA induces the silencing of homologous endogenous genes. Some aspects of RNAi appear to be conserved throughout evolution, including the processing of trigger dsRNAs into small 21-23-bp siRNAs and their use to guide the degradation of complementary mRNAs. Two remarkable features of RNAi were uncovered in plants and Caenorhabditid elegans. First, RNA-dependent RNA polymerase activities allow the synthesis of siRNA complementary to sequences upstream of or downstream from the initial trigger region in the target mRNA, leading to a transitive RNAi with sequences that had not been initially targeted. Secondly, systemic RNAi may cause the targeting of gene silencing in one tissue to spread to other tissues. Using transgenes expressing dsRNA, we investigated whether transitive and systemic RNAi occur in Drosophila. DsRNA-producing transgenes targeted RNAi to specific regions of alternative mRNA species of one gene without transitive effect directed to sequences downstream from or upstream of the initial trigger region. Moreover, specific expression of a dsRNA, using either cell-specific GAL4 drivers or random clonal activation of a GAL4 driver, mediated a cell-autonomous RNAi. Together, our results provide evidence that transitive and systemic aspects of RNAi are not conserved in Drosophila and demonstrate that dsRNA-producing transgenes allow powerful reverse genetic approaches to be conducted in this model organism, by knocking down gene functions at the resolution of a single-cell type and of a single isoform.

Animals↗
PubMed2004

Analysis of gene function in Trypanosoma brucei using RNA interference.

Trypanosoma brucei, a flagellate protozoa of the family Trypanosomatidae, has become one of the model systems for unicellular pathogens to study fundamentally important biological phenomena. The method of choice today to examine gene function in these organisms is RNA interference (RNAi). Messenger RNA (mRNA) degradation is triggered by double-stranded RNA (dsRNA) produced in vivo from transgenes transcribed from opposing tetracycline (tet)-inducible T7 RNA polymerase promoters, or hairpin RNA transcribed from the tet-inducible procyclic acidic repetitive protein promoter. This chapter describes some of the methods we employ for ablation of gene expression by RNAi in T. brucei with particular emphasis on transfection and cloning of procyclic cells, induction of dsRNA expression, isolation of RNA, and analysis of dsRNA and target mRNA.

Animals↗
PubMed2004

Argonaute2 is the catalytic engine of mammalian RNAi.

Gene silencing through RNA interference (RNAi) is carried out by RISC, the RNA-induced silencing complex. RISC contains two signature components, small interfering RNAs (siRNAs) and Argonaute family proteins. Here, we show that the multiple Argonaute proteins present in mammals are both biologically and biochemically distinct, with a single mammalian family member, Argonaute2, being responsible for messenger RNA cleavage activity. This protein is essential for mouse development, and cells lacking Argonaute2 are unable to mount an experimental response to siRNAs. Mutations within a cryptic ribonuclease H domain within Argonaute2, as identified by comparison with the structure of an archeal Argonaute protein, inactivate RISC. Thus, our evidence supports a model in which Argonaute contributes "Slicer" activity to RISC, providing the catalytic engine for RNAi.

Amino Acid Sequence↗
PubMed2006

A long-distance relationship between RNAi and Polycomb.

RNA interference (RNAi) pathways can result in sequence-specific transcriptional gene silencing on the level of chromatin. In this issue of Cell, Grimaud et al. (2006) reveal that the RNAi machinery is required for long-distance physical interactions between chromosomes mediated by the Polycomb repressive complex. These results suggest that the RNAi machinery may regulate higher-order nuclear organization.

Cell Nucleus↗
PubMed2004

OptiRNAi, an RNAi design tool.

RNA interference (RNAi), a recently developed reverse genetics tool, has many advantages compared to traditional gene knockout methods. Appropriate selection of double stranded RNAs identical to a specific region(s) of the target gene is critical for the successful implementation of this technology. Recently, Elbashir et al. [Methods 26 (2002) 199] has established empirical criteria for siRNA sequence selection that significantly improved the success rate for RNAi attempts. We have developed OptiRNAi, a computational tool, which uses the Elbashir et al. criteria to predict appropriate target sequences for siRNA production. Specificity of these siRNAs for the target of interest can then be assessed by the investigator using the embedded Blast search engine optimized for RNAi design. Thus, OptiRNAi is an efficient and user friendly tool for RNAi design based on criteria that are more stringent than other available tools.

Animals↗
PubMed2004

Enzymatic production of RNAi libraries from cDNAs.

RNA interference (RNAi) induced by small interfering (siRNA) or short hairpin RNA (shRNA) is an important research approach in mammalian genetics. Here we describe a technology called enzymatic production of RNAi library (EPRIL) by which cDNAs are converted by a sequence of enzymatic treatments into an RNAi library consisting of a vast array of different shRNA expression constructs. We applied EPRIL to a single cDNA source and prepared an RNAi library consisting of shRNA constructs with various RNAi efficiencies. High-throughput screening allowed us to rapidly identify the best shRNA constructs from the library. We also describe a new selection scheme using the thymidine kinase gene for obtaining efficient shRNA constructs. Furthermore, we show that EPRIL can be applied to constructing an RNAi library from a cDNA library, providing a basis for future whole-genome phenotypic screening of genes.

Calcium-Binding Proteins↗
PubMed2004

Visualizing a correlation between siRNA localization, cellular uptake, and RNAi in living cells.

RNA interference (RNAi) is the process by which short-interfering RNA (siRNA) target a specific mRNA for degradation through interactions with an RNA-induced silencing complex (RISC). Here, a clear correlation between siRNA localization, cellular uptake, and RNAi activity was discovered by delivering siRNA into cells using siRNA-TAT(47-57) peptide, siRNA-TAT(47-57)-derived oligocarbamate conjugates, or nanoparticles. For successful RNAi, the localization of siRNA was distinctly perinuclear, suggesting that siRNA is targeted to these regions for interactions with RISC to induce RNAi. siRNA sequence variation and the presence of the target mRNA apparently did not change the subcellular localization pattern of siRNA. Intriguingly, siRNA conjugated to TAT(47-57) peptide or TAT(47-57)-derived oligocarbamate resulted in efficient RNAi activity and perinuclear localization of siRNA that was distinctly different from nonconjugated free TAT peptide nucleolar localization. These results suggest that interactions with RISC dictate siRNA localization even when siRNA is conjugated to TAT(47-57) peptide.

Base Sequence↗
PubMed2006

Distinct populations of primary and secondary effectors during RNAi in C. elegans.

RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: "Primary siRNAs" (derived from DICER nuclease-mediated cleavage of the original trigger) and "secondary siRNAs" [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.

Animals↗
PubMed2006

RNAi-mediated downregulation of urokinase plasminogen activator and its receptor in human meningioma cells inhibits tumor invasion and growth.

In recent years, RNA interference (RNAi) has emerged as an effective method to target specific genes for silencing. Several groups are actively exploring the use of small interfering RNA (siRNA) for therapeutic applications to treat cancer. Our previous studies have demonstrated the inhibition of various proteases, including serine proteases, cysteine proteases and matrix metalloproteases, via RNA interference (RNAi) in gliomas. Similar to gliomas, malignant meningiomas also exhibit elevated protease levels in comparison to normal brain and benign meningiomas. Here, we used siRNA to simultaneously target urokinase plasminogen activator (uPA) and its receptor, uPAR. A human CMV promoter-driven mammalian expression vector (pU2) was used to produce hairpin double-stranded RNA (hp RNA) to target uPA and uPAR. As determined by Western blotting and fibrin zymography, pU2 effectively inhibited uPAR protein levels and uPA enzymatic activity in meningioma cells (IOMM-Lee). In vitro studies (Matrigel invasion and spheroid migration) revealed reduced meningioma cell invasion and migration. Intratumoral injections of the plasmid vector expressing siRNA for uPA and uPAR resulted in regression of pre-established, subcutaneous tumors in mice. In addition, in vivo studies of mice injected with pU2-transfected meningioma cells revealed inhibition of intracranial tumor formation. These findings suggest that siRNA can be used as a potent and specific therapeutic tool for the treatment of malignant meningiomas in humans.

Animals↗
PubMed2005

RNAi mechanisms in Caenorhabditis elegans.

RNA interference (RNAi) is a form of gene silencing induced by double stranded RNA (dsRNA) that is processed into short interfering RNAs (siRNAs). RNAi can induce both post-transcriptional and transcriptional gene silencing. In Caenorhabditis elegans, there are several distinct pathways where post-transcriptional or/and transcriptional RNAi mechanisms are involved. RNAi in C. elegans is also systemic and heritable. This review will discuss RNAi related pathways, features of RNAi in C. elegans and possibilities of endogenous gene regulation by RNAi.

Animals↗
PubMed2001

RNA interference of gene expression (RNAi) in cultured Drosophila cells.

RNA interference (RNAi) can be used to silence genes in a number of taxa, including plants, nematodes, protozoans, flies, and mammals represented by mouse embryos and cultured mammalian cells. To investigate signal transduction pathways, we used RNAi on Drosophila-cultured cells, which affords the opportunity to study protein function in a simple, well-defined cell culture system. Furthermore, the results obtained from experiments performed on cultured cells can be confirmed and extended in the whole organism, which, in the case of Drosophila, is also RNAi responsive. RNAi takes advantage of the unique ability of double-stranded RNA (dsRNA) molecules to induce posttranscriptional gene silencing in a highly specific manner. This silencing is efficacious and long-lived, as it is passed to subsequent generations in insect cell culture. To date, all Drosophila cell lines tested (S2, KC, BG2-C6, and Shi) respond to dsRNAs by ablating expression of the target protein. Furthermore, all dsRNAs tested (more than 15) have been effective at silencing the target gene. Drosophila cell cultures are simple, easily manipulated model systems that will facilitate loss-of-function studies applicable to a wide variety of questions.

Animals↗
PubMed2006

Enzymatically prepared RNAi libraries.

Large-scale RNA interference (RNAi) screens in mammalian cells have mainly used synthetic small interfering RNA (siRNA) or short hairpin RNA (shRNA) libraries. The RNAi triggers for both of these approaches were designed with algorithm-based predictions to identify single sequences for mRNA knockdown. Alternatives to these approaches have recently been developed using enzymatic methods. Here we describe the concepts of enzymatically prepared shRNA and siRNA libraries, and discuss their strengths and limitations.

Animals↗