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At least 1,135 records · Page 63Linked to original sources
PubMed2004

RNA interference by osmotic lysis of pinosomes: liposome-independent transfection of siRNAs into mammalian cells.

The osmotic lysis of pinosomes procedure has been adapted to deliver small interfering RNAs (siRNAs) into cells in culture. Under hypertonic conditions, siRNAs were internalized into pinosomes. A subsequent osmotic shock in hypotonic buffer disrupted the pinosomes and caused the release of siRNAs into the cell cytoplasm. Both steps could be demonstrated directly using fluorescein-labeled siRNAs and confocal laser-scanning microscopy. Uptake by the pinocytosis/osmotic lysis procedure is concentration- and time-dependent. At an siRNA concentration of 0.4 microM, treatment for 40 or 80 min results in silencing efficiencies of 60% and 90%, respectively, after 44 h. A double treatment resulted in approximately equal silencing efficiencies but in reduced viability. This method has been used on a variety of human and murine cell lines including HEK293, HeLa SS6, and SW3T3 cells. Targets such as lamin A/C and Eg5 were effectively silenced. Novel silencing data are provided for Ki67, one of the few reliable prognostic markers for tumor patients. The new procedure avoids certain technical problems encountered with commercial transfection reagents while yielding silencing efficiencies that are comparable to those obtained with liposome-mediated siRNA transfection.

3T3 Cells↗
PubMed2006

Microarray analysis of gene-expression profile in hepatocellular carcinoma cell, BEL-7402, with stable suppression of hLRH-1 via a DNA vector-based RNA interference.

To establish a cell line with a permanent suppression of hLRH-1 in this study, a stable RNAi vector (pSineohLRH-1) targeting hLRH-1 was constructed and introduced into hepatocellular carcinoma cell, BEL-7402. By semiquantitative RT-PCR analysis, the expression of hLRH-1 in BEL-7402 cells carrying pSineohLRH-1 was shown to be significantly suppressed by up to approximately 60%. In addition, microarray analysis was carried out to assess the extent of altered gene expression in BEL-7402 cells with stable knockdown of hLRH-1. Direct comparison of gene-expression profiles of more than 18,000 genes showed that 405 of the expressed genes in hLRH-1-knockdown cells differed dramatically in expression levels from those in controls, which suggested the even extensive biological functions of hLRH-1. Interestingly, among those differentially expressed genes, some are cancer-associated such as Gadd45beta and PTEN, and their expressions were further validated. Although the identification of the exact relationship between these genes and hLRH-1 awaits intensive investigation, the findings of this study provide new insights into the mechanism by which hLRH-1 is involved in tumorigenesis.

Carcinoma, Hepatocellular↗
PubMed2005

Two novel proteins, dos1 and dos2, interact with rik1 to regulate heterochromatic RNA interference and histone modification.

BACKGROUND: Chromosomal behavior during mitosis and meiosis depends in part on heterochromatic modifications such as histone H3 lysine-9 methylation (H3K9me). In fission yeast, the Heterochromatin Protein 1 homolog Swi6 recognizes H3K9me, silences transcription, and retains cohesin at pericentromeric repeats. Heterochromatin formation also depends on processing of transcripts derived from centromeric repeats by the RNAi machinery. The DDB1 homolog, Rik1, and histone methyltransferase, Clr4, act in a complex to promote H3K9me. However, the mechanism underlying this interaction is poorly understood. RESULTS: Using a cytological screen, we have identified two novel genes, dos1(+) and dos2(+), which are required for localization of Swi6. Deletion of either of these genes results in mitotic and meiotic chromosome missegregation, defects in mitotic centromeric cohesion and meiotic telomere clustering, and loss of heterochromatic silencing. Dos1 is predominantly located in the nucleus in a Dos2-dependent manner and directly interacts with Rik1. Each of these genes is required for the association of H3K9me with centromeric repeats, as well as for the production of small interfering RNAs. CONCLUSIONS: Dos1 and Dos2 are required for the formation of heterochromatin in fission yeast. We hypothesize that the physical interaction between Dos1 and Rik1 represents a role in regulating activity of the Rik1/Clr4 complex. Dos2 contributes to this role by regulating Dos1 localization. Our findings suggest a mechanism for recruitment of Clr4 in the RNAi-dependent heterochromatin pathway, in which Dos1 and Dos2 are essential.

Carrier Proteins↗
PubMed2005

RNA interference of Arabidopsis beta-amylase8 prevents maltose accumulation upon cold shock and increases sensitivity of PSII photochemical efficiency to freezing stress.

It has been suggested that beta-amylase (BMY) induction during temperature stress in Arabidopsis could lead to starch-dependent maltose accumulation, and that maltose may contribute to protection of the electron transport chain and proteins in the chloroplast stroma during acute stress. A time-course transcript profiling analysis for cold shock at 4 degrees C revealed that BMY8 (At4g17090) was induced specifically in response to cold shock, while major induction was not observed for any of the other eight beta-amylases. A parallel metabolite-profiling analysis revealed a robust transient maltose accumulation during cold shock. BMY8 RNAi lines with lower BMY8 expression exhibited a starch-excess phenotype, and a dramatic decrease in maltose accumulation during a 6-h cold shock at 4 degrees C. The decreased maltose content was also accompanied by decreased glucose, fructose and sucrose content in the BMY8 RNAi plants, consistent with the roles of beta-amylase and maltose in transitory starch metabolism. BMY8 RNAi lines with reduced soluble sugar content exhibited diminished chlorophyll fluorescence as F(v)/F(m) ratio compared with wild type, suggesting that PSII photochemical efficiency was more sensitive to freezing stress. Together, carbohydrate analysis and freezing stress results of BMY8 RNAi lines indicate that increased maltose content, by itself or together through a maltose-dependent increase in other soluble sugars, contributes to the protection of the photosynthetic electron transport chain during freezing stress.

Arabidopsis↗
PubMed2006

Adapting rice anther culture to gene transformation and RNA interference.

Anther culture offers a rapid method of generating homozygous lines for breeding program and genetic analysis. To produce homozygous transgenic lines of rice (Oryza sativa L.) in one step, we developed an efficient protocol of anther-callus-based transformation mediated by Agrobacterium after optimizing several factors influencing efficient transformation, including callus induction and Agrobacterium density for co-cultivation. Using this protocol, we obtained 145 independent green transformants from five cultivars of japonica rice by transformation with a binary vector pCXK1301 bearing the rice gene, Xa21 for resistance to bacterial blight, of which 140 were further confirmed by PCR and Southern hybridization analysis, including haploids (32.1%), diploids (62.1%) and mixoploids (7.5%). Fifteen diploids were found to be doubled haploids, which accounted for 10.7% of the total positive lines. Finally, by including 28 from colchicine induced or spontaneous diploidization of haploids later after transformation, a total of 43 doubled haploids (30.7%) of Xa21 transgenic lines were obtained. We also generated two RNAi transgenic haploids of the rice OsMADS2 gene, a putative redundant gene of OsMADS4 based on their sequence similarity, to investigate its possible roles in rice flower development by this method. Flowers from the two OsMADS2 RNAi transgenic haploids displayed obvious homeotic alternations, in which lodicules were transformed into palea/lemma-like tissues, whereas identities of other floral organs were maintained. The phenotypic alternations were proved to result from specific transcriptional suppression of OsMADS2 gene by the introduced RNAi transgene. The results confirmed that OsMADS2 is involved in lodicule development of rice flower and functionally redundant with OsMADS4 gene. Our results demonstrated that rice anther culture could be adapted to gene transformation and RNAi analysis in rice.

Adaptation, Biological↗
PubMed2006

[Inhibition of hepatitis B virus replication and expression by RNA interference in vivo].

OBJECTIVE: To evaluate the inhibitory effect of small interfering RNA (siRNA) targeting HBV C gene region on hepatitis B virus (HBV) in vivo. METHODS: An animal model of HBV infection was developed hydrodynamically, and pcDNA3.1-HBV and siRNA were together injected into the tail vein of the BALB/c mice. HBsAg was analyzed by time-resolved immunofluorometric assay, HBV DNA was analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR, and viral specific proteins (HBsAg and HBcAg) in the mice livers were assayed using immunohistochemical staining. RESULTS: In the mice, the siRNA effectively inhibited HBV replication and expression compared with the controls. The inhibitive effect of siRNA on HBV lasted at least 3 days. CONCLUSION: These results demonstrate that RNAi can substantially inhibit HBV replication and expression in vivo.

Animals↗
PubMed2002

Selective silencing of viral gene expression in HPV-positive human cervical carcinoma cells treated with siRNA, a primer of RNA interference.

Selective silencing of mammalian gene expression has recently been achieved using short interfering RNA (siRNA). Synthetic siRNA targets homologous mRNA for degradation and the process is highly efficient. Here we demonstrate siRNA silencing of pathogenic viral gene expression. As a well characterized model we chose cervical carcinoma cells positive for human papillomavirus type 16. Over 90% of human cervical cancers are positive for papillomavirus and abnormal cell proliferation is driven by co-operative effects of viral E6 and E7 genes. We sought to silence HPV E6 and E7 gene expression using siRNAs to target the respective viral mRNAs. Our results indicate selective degradation of E6 and E7 mRNAs. Silencing was sustained for at least 4 days following a single dose of siRNA. E6 silencing induced accumulation of cellular p53 protein, transactivation of the cell cycle control p21 gene and reduced cell growth. In contrast, E7 silencing induced apoptotic cell death. HPV-negative cells appeared unaffected by the anti-viral siRNAs. Thus we demonstrate for the first time (i) that siRNA can induce selective silencing of exogenous viral genes in mammalian cells, and (ii) that the process of siRNA interference does not interfere with the recovery of cellular regulatory systems previously inhibited by viral gene expression.

Apoptosis↗
PubMed2005

Isolation of white gene orthologue of the sawfly, Athalia rosae (Hymenoptera) and its functional analysis using RNA interference.

We isolated and characterized the white gene orthologue of the sawfly, Athalia rosae (Hymenoptera). The A. rosae white (Ar white) cDNA cloned was 2058-bp long encoding 685 amino acids in a single open reading frame (ORF). Comparison of the cDNA sequence with the genomic DNA sequence revealed that the ORF was derived from 11 exons. Ar white was a single copy gene as evidenced by genomic Southern blotting and its cytological localization on the metaphase chromosomes. The deduced amino acid sequence aligned well with known insect white orthologous gene products sharing conserved regions such as the ATP-binding motif and the six transmembrane-spanning segments. Expression of Ar white was detected at embryonic and pupal stages by Northern blotting. In situ hybridization detected the embryonic expression in a pair of the lateral tips of protocephalic placodes from where optic organs are formed. Ar white function was examined using double-stranded RNA (dsRNA)-mediated interference. The synthesized dsRNA targeting Ar white transcripts caused a decrease in the level of the original mRNAs, and resulted in the white phenocopy in the embryonic eye pigmentation when microinjected into eggs from wild-type females. The effects occurred in a dose-dependent manner.

ATP-Binding Cassette Transporters↗
PubMed2006

RNA interference reveals a differential role of FAK and Pyk2 in cell migration, leading edge formation and increase in focal adhesions induced by LPA in intestinal epithelial cells.

In the gastrointestinal mucosa, cell migration plays a crucial role in the organization and maintenance of tissue integrity but the mechanisms involved remain incompletely understood. Here, we used small-interfering RNA (siRNA)-mediated depletion of focal adhesion kinase (FAK) protein to determine the role of FAK in wound-induced migration and cytoskeletal organization in the non-transformed intestinal epithelial cells IEC-6 and IEC-18 stimulated with the G protein-coupled receptors (GPCR) agonist lysophosphatidic acid (LPA). Treatment of these cells with FAK siRNA substantially reduced FAK expression, but did not affect the expression of proline-rich tyrosine kinase 2 (Pyk2). Knockdown of FAK protein significantly inhibited LPA-induced migration of both IEC-18 and IEC-6 cells. LPA induced reorganization of actin and microtubule cytoskeleton in the leading edge was largely inhibited in FAK siRNA-transfected IEC-18 cells. Interestingly, in contrast to the FAK-/- cells, which exhibit an increased number of prominent focal adhesions when plated on fibronectin, FAK knockdown IEC-18 cells exhibited dramatically decreased number of focal adhesions in response to both LPA and fibronectin as compared with the control cells. We also used siRNAs to knockdown Pyk2 expression without reducing FAK expression. Depletion of Pyk2 did not prevent LPA-induced migration or cytoskeletal reorganization in IEC-18 cells. In conclusion, our study shows that FAK plays a critical role in LPA-induced migration, cytoskeletal reorganization, and assembly of focal adhesions in intestinal epithelial cells whereas depletion of Pyk2 did not interfere with any of these responses elicited by LPA.

Cell Line↗
PubMed2003

Use of RNA interference to inhibit integrin (alpha6beta4)-mediated invasion and migration of breast carcinoma cells.

The application of small interfering RNA (siRNA) oligonucleotides to silence gene expression has profound implications for the intervention of human diseases including cancer. Using this technique, we explored the possibility that the alpha6beta4 integrin, a laminin adhesion receptor with a recognized role in the invasive phenotype of many carcinomas, represents a potential therapeutic target to inhibit the migration and invasion of carcinoma cells. We found that siRNA oligonucleotides targeted to either subunit of the alpha6beta4 integrin reduced cell surface expression of this integrin and resulted in decreased invasion of MDA-MB-231 breast carcinoma cells. Interestingly, reduced alpha6beta4 expression also promoted decreased migration on non-laminin substrata indicating that this integrin can function in a ligand-independent manner. In addition, the absence of beta4 expression in these cells augmented the formation of alpha6beta1 heterodimers and increased adhesion to laminin-1. Taken together, these results substantiate the importance of the alpha6beta4 integrin in invasion and migration that has been demonstrated previously by expression of the beta4 subunit in beta4-deficient cell lines and by function blocking antibodies. Furthermore, these data suggest that the utilization of siRNA oligonucleotides to reduce the expression of the alpha6beta4 integrin may be a useful approach to prevent carcinoma cell progression.

Biotinylation↗
PubMed2003

Use of RNA interference-mediated gene silencing and adenoviral overexpression to elucidate the roles of AKT/protein kinase B isoforms in insulin actions.

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.

3T3 Cells↗
PubMed2001

Genes and mechanisms related to RNA interference regulate expression of the small temporal RNAs that control C. elegans developmental timing.

RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Here we show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation.

Animals↗