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At least 1,099 records · Page 61Linked to original sources
PubMed2005

Blockage of the macrophage migration inhibitory factor expression by short interference RNA inhibited the rejection of an allogeneic tracheal graft.

We investigated the inhibitory effect of blocking the macrophage migration inhibitory factor (MIF) on the fibrous obstruction of a transplanted allograft in a murine model of obstructive bronchiolitis (OB). Tracheal grafts from C57BL/6 mice were transplanted into a subcutaneous pouch of BALB/c. Three days after transplantation, liposome including short interference (si) RNA for MIF was injected into the lumen of the grafts. The allografts were then harvested 7, 14 or 28 days after transplantation for an evaluation of the morphological changes. The MIF expression, which was ubiquitously recognized in the epithelium of allografts, decreased after the in vivo transfection of MIF siRNA. OB formation was therefore inhibited significantly more by the treatment with MIF siRNA than the allografts injected with empty liposome on the 14th day, however, no difference was observed between them on the 28th day. Treatment with MIF siRNA inhibits the destruction of tracheal allografts and OB formation in the early phase, and MIF was thus found to be one of the major cytokines involved in the rejection of the allogeneic trachea.

Animals↗
PubMed2006

The effect of silencing NKG2D through RNA interference on receptor functions in interleukin-2-activated human natural killer cells.

Natural killer (NK) cells are effectors of the innate immunity involved in tumor surveillance. NKG2D is a potent activating receptor eliciting cytokine and cytolytic NK responses upon recognition of tumor-associated ligands. We engineered primary interleukin (IL)-2-activated human NK cells to express constitutively low levels of NKG2D by lentiviral delivery of small interfering RNA. NKG2D-mediated effector functions were strongly impaired in NKG2D(low) NK cells. Reduction of NKG2D surface expression to 15%, corresponding to receptor levels in resting NK cells, rendered cells fully insensitive to NKG2D triggering. These data underscore the importance of NKG2D receptor cell surface density and suggest a threshold of expression for optimal reactivity of human NK cells.

Cells, Cultured↗
PubMed2005

RNA interference against a glioma-derived allele of EGFR induces blockade at G2M.

Amplification and mutation of the epidermal growth factor receptor (EGFR) is common in astrocytoma. The most frequently occurring mutation (DeltaEGFR, EGFRvIII) deletes exons 2-7 from this receptor tyrosine kinase (RTK), and signals constitutively in the absence of ligand. DeltaEGFR is not found in normal tissue, and therefore represents an attractive therapeutic target. Here, we show that a small interfering RNA (siRNA) directed against the unique exon 1/exon 8 junction sequence of DeltaEGFR efficiently suppressed expression of DeltaEGFR in rodent fibroblasts and in two human glioblastoma cell lines. SiRNA-mediated depletion of DeltaEGFR led to reduction in the levels of phosphorylated Akt in glioma cells, was associated with increased apoptosis, and induced partial arrest at the G2M phase of the cell cycle. Inhibitors of PI3 kinase cooperated with siRNA treatment, leading to further increases in both cell cycle blockade and apoptosis. Importantly, cell cycle blockade could be reversed, and apoptosis rescued using a conditional allele of Akt, implicating Akt as a primary target of combination therapy. This study demonstrates the therapeutic potential of siRNA to impact DeltaEGFR as a glioma-specific target, and offers a mechanistic rationale for combining siRNA and small molecule inhibitor therapies against distinct components in the EGFR signaling pathway.

Astrocytoma↗
PubMed2006

RNA interference (RNAI) as a tool to engineer high nutritional value in chicory (Chicorium intybus).

The major component of chicory (Chicorium intybus) root is inulin, which is a polymer of fructose. Inulin production from chicory is hampered by the enzyme fructan 1-exohydrolase (1-FEH) that degrades inulin and limits its yield. Increased FEH activity results in massive breakdown of fructan and production of Fructose and inulo-n-oses. The latter phenomena are to be avoided for industrial fructan production. RNA silencing, which is termed post-transcriptional gene silencing (PTGS) in plants, is an RNA degradation process through sequence specific nucleotide interactions induced by double-stranded RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and co-suppression, in terms of its efficiency and stability. We are generating a transgenic chicory plants with suppressed FEH (exohydrolas) genes using RNAi resulting in supressed inulin degradation. A small but important part of the construct is a sequence unique for the target gene (exons) or genes,which were cloned. The hairpin constructs were made and chicory was transformed by Agrobacterium tumifaciense, strain (C58C1). The transgenics should be select and check by means of molecular techniques.

Carbohydrate Metabolism↗
PubMed1999

The rde-1 gene, RNA interference, and transposon silencing in C. elegans.

Double-stranded (ds) RNA can induce sequence-specific inhibition of gene function in several organisms. However, both the mechanism and the physiological role of the interference process remain mysterious. In order to study the interference process, we have selected C. elegans mutants resistant to dsRNA-mediated interference (RNAi). Two loci, rde-1 and rde-4, are defined by mutants strongly resistant to RNAi but with no obvious defects in growth or development. We show that rde-1 is a member of the piwi/sting/argonaute/zwille/eIF2C gene family conserved from plants to vertebrates. Interestingly, several, but not all, RNAi-deficient strains exhibit mobilization of the endogenous transposons. We discuss implications for the mechanism of RNAi and the possibility that one natural function of RNAi is transposon silencing.

Amino Acid Sequence↗
PubMed2006

[The influence of downregulation of ezrin expression by RNA interference on the growth and metastasis of hepatocellular carcinoma: experiment in vitro].

OBJECTIVE: To investigate the effect of membrane-cytoskeleton linker ezrin on the growth and metastasis of human hepatocellular carcinoma cell (HCC) lines. METHODS: Human HCC cells of the lines SF/SMMC7721, MHCC97-H, MHCC-1, and HepG2 were cultured. Four pairs of small interfering RNA (siRNA) targeting erzin were designed and transfected into the HCC cells. 48 h after transfection the cell total RNA was extracted and 72 h later the total cell protein was extracted. RT-PCR and Western blotting were used to detect the transfection rates so as to screen the most effective siRNA to be transfected into the HCC cells. HCC cells were collected every day for 7 days to extract the total RNA and protein. Real-time PCR and Western blotting were used to detect the downregulation rate of erzin at different times. MTT method was used to detect the proliferation of the cells. Flow cytometry was used to detect the cell cycle and apoptosis. Scanning electron microscopy was used to observe the cell pseudopods. Transwell test was used to detect the invasion ability of the transfected HCC cells. RESULTS: Real-time PCR and western-blotting revealed that ezrin siRNA notably down-regulated ezrin expression at both mRNA and protein levels. Down-regulation of ezrin expression distinctly decreased the proliferation rates of these 4 kinds of HCC line. After RNAi treatment the cell proportion in G(2)-M phase decreased from 28.07% to 21.53% in the SF/SMMC7721 cells, from 24.94% to 13.92% in the MHCC97-H cells, from 19.30% to 13.2% in the MHCC-1cells, and from 7.73% to 4.24% in the HepG2 cells. After RNAi treatment, the number of pseudopods decreased from 20.8 +/- 3.0 to 13.2 +/- 2.4 in the SF/SMMC7721: cells (P < 0.05), from 18.4 +/- 2.7 to 14.0 +/- 2.9 in the MHCC97-H cells (P < 0.01), from 22.6 +/- 3.5 to 13.3 +/- 1.9 in the MHCC-1: cells (P < 0.01), and from 31.0 +/- 2.9 to 17.8 +/- 2.3 in the HepG2 cells (P < 0.01); and the motility and invasiveness decreased from 49.9 +/- 7.7 to 31.9 +/- 5.2 in the SF/SMMC7721 cells (P < 0.05), from 58.5 +/- 4.2 to 33.0 +/- 3.3 in the MHCC97-H cells (P < 0.01), from 57.6 +/- 6.1 to 28.3 +/- 3.4 in the MHCC-1 cells (P < 0.01), and from 37.3 +/- 3.0 to 25.3 +/- 2.3 in the HepG2 cells (P < 0.01). The pseudopods of the HCC cells remarkably shortened and decreased in number (for the SF/SMMC7721 cells: t = 4.95, P < 0.05, for the MHCC97-H cells: t = 5.88, P < 0.01, for the MHCC-1 cells: t = 5.56, P < 0.01, and for the HepG2 cells: t = 5.71, P < 0.01) after siRNA interference. CONCLUSION: Ezrin is necessary for HCC proliferation and invasion. It is probably an important factor to inhibit tumor reoccurrence and metastasis.

Blotting, Western↗
PubMed2000

Double-stranded RNA interference shows that Engrailed controls the synaptic specificity of identified sensory neurons.

The transcription factor Engrailed (En) controls the topography of axonal projections by regulating the expression of cell-adhesion molecules [1] [2] [3] [4] but it is not known whether it also controls the choice of individual synaptic target cells. In the cercal sensory system of the larval cockroach (Periplaneta americana), small numbers of identified wind-sensitive sensory neurons form highly specific synaptic connections with 14 identified giant interneurons [5] [6], and target-cell choice is independent of the pattern of axonal projections [6]. En is a putative positional determinant in the array of cercal sensory neurons [7]. In the present study, double-stranded RNA (dsRNA) interference [8] was used to abolish En expression. This treatment changed the axonal arborisation and synaptic outputs of an identified En-positive sensory neuron so that it came to resemble a nearby En-negative cell, which was itself unaffected. We thus demonstrate directly that En controls synaptic choice, as well as axon projections.

Animals↗
PubMed2004

Lack of impact of the loss of constitutive folate receptor alpha expression, achieved by RNA Interference, on the activity of the new generation antifolate pemetrexed in HeLa cells.

Pemetrexed [PMX (Alimta)] is a new generation antifolate with activity in a variety of solid tumors. It is an excellent substrate for most folate transporters, notably the reduced folate carrier (RFC) and folate receptor (FR)-alpha. The role of FR-alpha in PMX pharmacological activity is uncertain. Whereas high-level expression may enhance the activity of this agent, it is not clear what role constitutive levels of this transporter contribute to PMX activity. In this study, constitutive levels of FR-alpha expression were abolished by small interfering RNA-induced silencing in HeLa cells and RFC-null HeLa R5 cells as confirmed by Northern blotting, immunohistochemistry, and cell surface binding. PMX growth inhibition was unchanged in HeLa and R5 cells in the absence of FR-alpha expression. Loss of FR-alpha expression did not decrease net accumulation of PMX in either wild-type or RFC-null HeLa cells. Likewise, folate pools in wild-type HeLa cells were not decreased by FR-alpha gene silencing and were negligibly affected in the RFC-null R5 subline grown with 5-formyltetrahydrofolate. FR-alpha surface binding in HeLa cells was shown to be greater than that in a variety of other human solid tumor cell lines. Hence, constitutively expressed FR-alpha in HeLa cells does not contribute to PMX activity in the presence or absence of RFC function. This is likely the case in many human solid tumor cell lines.

Apoptosis↗
PubMed2005

RNA interference targeting leptin gene effect on hepatic stellate cells.

To construct the specific siRNA expression vectors and investigate their effect on leptin and collagen I in HSC, which provide a new approach to the prevent and treat hepatic fibrosis. The five siRNAs against leptin gene were transcript synthesized intracellularly by expression templates of plasmid vector psiRNA-hH1neo. The recombinant leptin siRNA plasmid vectors could express in eukaryocyte, and then to evaluate them by using enzyme cutting and sequencing. The recombinant plasmids were transfected into HSCs using Lipofectamine methods respectively. The cells were selected after growing in DMEM containing 300 microg/mL G418 for about 4 weeks. Gene expression of leptin and collagen I were showed by Western blot analysis and reverse transcription polymerase chain reaction (RT-PCR). Identification by enzyme cutting and sequencing showed that the leptin siRNA expression vectors were constructed successfully, and leptin siRNA could inhibit the leptin and collagen I gene expression effectively. It was concluded that RNA interference-mediated silencing of leptin gene diminished leptin and collagen I gene expression in HSCs. Furthermore, attenuated the extracellular matrix over-deposition at the same time. Leptin gene is ideal targets of gene therapy for liver fibrosis.

Cell Line↗