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Results for “RNA interference”

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PubMed2004

Modulation of nuclear steroid receptors by ursodeoxycholic acid inhibits TGF-beta1-induced E2F-1/p53-mediated apoptosis of rat hepatocytes.

We have recently shown that both ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) prevent transforming growth factor beta1 (TGF-beta1)-induced hepatocyte apoptosis by modulating the E2F-1/p53/Bax pathway. In addition, activation of glucocorticoid (GR) and mineralocorticoid receptors (MR) inhibits apoptosis in various systems. UDCA induces a ligand-independent activation of the GR, thus potentially regulating a number of targets. In this study, we investigated the role of GR and MR during TGF-beta1-induced hepatocyte apoptosis, and identified additional antiapoptotic targets for UDCA. Our results showed that in primary hepatocytes, TGF-beta1 induced 40-50% decreases in gr and mr mRNA expression (p < 0.01), together with up to 10-fold reductions in their protein levels (p < 0.01). Notably, pretreatment with UDCA resulted in a significant upregulation of nuclear steroid receptors (p < 0.05), which coincided with 2- and 3-fold increases in the level of GR and MR nuclear translocation, respectively, when compared with that of TGF-beta1 alone (p < 0.05). Similarly, TUDCA induced GR and MR nuclear translocations (p < 0.05) and markedly prevented MR protein changes associated with TGF-beta1 (p < 0.05) without affecting GR protein levels. Moreover, when interference RNA was used to inhibit GR and MR, UDCA no longer protected hepatocytes against TGF-beta1-induced apoptosis. In fact, the protective effect of UDCA in TGF-beta1-associated caspase activation decreased from 65 to <10% when GR or MR function was blocked. Finally, the TGF-beta1-induced E2F-1/Mdm-2/p53 apoptotic pathway, normally inhibited by UDCA, was not regulated by the bile acid after GR or MR silencing. These results demonstrate that UDCA protects against apoptosis through an additional pathway that involves nuclear receptors GR and MR as key factors. Further, the E2F-1/Mdm-2/p53 apoptotic pathway appears to be a prime target for UDCA-induced steroid receptor activation.

Active Transport, Cell Nucleus↗

PubMed2004

Helicobacter pylori CagA induces Ras-independent morphogenetic response through SHP-2 recruitment and activation.

The CagA protein of Helicobacter pylori, which is injected from the bacteria into bacteria-attached gastric epithelial cells, is associated with gastric carcinoma. CagA is tyrosine-phosphorylated by Src family kinases, binds the SH2 domain-containing SHP-2 phosphatase in a tyrosine phosphorylation-dependent manner, and deregulates its enzymatic activity. We established AGS human gastric epithelial cells that inducibly express wild-type or a phosphorylation-resistant CagA, in which tyrosine residues constituting the EPIYA motifs were substituted with alanines. Upon induction, wild-type CagA, but not the mutant CagA, elicited strong elongation of cell shape, termed the "hummingbird" phenotype. Time-lapse video microscopic analysis revealed that the CagA-expressing cells exhibited a marked increase in cell motility with successive rounds of elongation-contraction processes. Inhibition of CagA phosphorylation by an Src kinase inhibitor, PP2, or knockdown of SHP-2 expression by small interference RNA (siRNA) abolished the CagA-mediated hummingbird phenotype. The morphogenetic activity of CagA also required Erk MAPK but was independent of Ras or Grb2. In AGS cells, CagA prolonged duration of Erk activation in response to serum stimulation. Conversely, inhibition of SHP-2 expression by siRNA abolished the sustained Erk activation. Thus, SHP-2 acts as a positive regulator of Erk activity in AGS cells. These results indicate that SHP-2 is involved in the Ras-independent modification of Erk signals that is necessary for the morphogenetic activity of CagA. Our work therefore suggests a key role of SHP-2 in the pathological activity of H. pylori virulence factor CagA.

Animals↗

PubMed2004

Chelation of cellular calcium modulates hypoxia-inducible gene expression through activation of hypoxia-inducible factor-1alpha.

Hypoxia-Inducible Factor-1 (HIF-1) is the key transcription factor in control of the expression of hypoxia-inducible genes needed by cells to adapt to decreased oxygen availability. Herein, we investigated the HIF-1alpha-mediated gene expression of carbonic anhydrase 9 (CA9) in response to hypoxia and changes of intracellular calcium levels in the neuroblastoma cell line SH-SY5Y. Decreasing the intracellular calcium level by BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) induced HIF-1alpha nuclear accumulation and enhanced HIF-1 DNA binding within 1 h of incubation. Like hypoxia, BAPTA stimulated HIF-1-dependent transcription by increasing the activity of the C-terminal transactivation domain of HIF-1alpha and greatly enhanced expression of the HIF-1 target gene CA9. Detailed analysis of HIF-1alpha accumulation revealed that BAPTA attenuated the interaction of HIF-1alpha with von-Hippel-Lindau protein thus decreasing proteasomal degradation of HIF-1alpha. Knock down of HIF-1alpha mRNA and protein by small interference RNA for HIF-1alpha revealed that both hypoxia and the BAPTA-induced gene expression of CA9 were strictly dependent on HIF-1alpha. In contrast, elevation of cytosolic calcium level by thapsigargin reduced the BAPTA-mediated effects. Measurements of intracellular calcium under hypoxia revealed a change in the cellular calcium distribution. BAPTA-dependent induction of HIF-1 activity was not caused by its in vitro capability to chelate iron. Instead, effective chelation of cellular calcium caused the accumulation of HIF-1alpha protein through inhibition of HIF-prolyl hydroxylases and activated HIF-1-dependent gene expression under normoxic conditions.

Blotting, Western↗

PubMed2005

Tumor necrosis factor alpha-induced desumoylation and cytoplasmic translocation of homeodomain-interacting protein kinase 1 are critical for apoptosis signal-regulating kinase 1-JNK/p38 activation.

The apoptosis signal-regulating kinase 1 (ASK1)-JNK/p38 signaling pathway is pivotal component in cell apoptosis and can be activated by a variety of death stimuli including tumor necrosis factor (TNF) alpha and oxidative stress (reactive oxygen species). However, the mechanism for ASK1 activation is not fully understood. We have recently identified ASK1-interacting protein (AIP1) as novel signal transducer in TNFalpha-induced ASK1 activation by facilitating dissociation of ASK1 from its inhibitor 14-3-3. In the present study, we employed yeast two-hybrid system using the N-terminal domain of AIP1 as bait and identified homeodomain-interacting protein kinase 1 (HIPK1) as an AIP1-associated protein. Interestingly, we showed that TNFalpha induced HIPK1 desumoylation concomitant with a translocation from nucleus to cytoplasm at 15 min followed by a return to nucleus by 60 min. The kinetics of HIPK1 translocation correlates with those of stress-induced ASK1-JNK/P38 activation. A specific JNK inhibitor blocked the reverse but not the initial translocation of HIPK1, suggesting that the initial translocation is an upstream event of ASK1-JNK/p38 signaling and JNK activation regulates the reverse translocation as a feedback mechanism. Consistently, expression of HIPK1 increased, whereas expression of a kinase-inactive form (HIPK1-D315N) or small interference RNA of HIPK1 decreased stress-induced ASK1-JNK/P38 activation without effects on IKK-NF-kappaB signaling. Moreover, a sumoylation-defective mutant of HIPK1 (KR5) localizes to the cytoplasm and is constitutively active in ASK1-JNK/P38 activation. Furthermore, HIPK1-KR5 induces dissociation of ASK1 from its inhibitors 14-3-3 and thioredoxin and synergizes with AIP1 to induce ASK1 activation. Our study suggests that TNFalpha-induced desumoylation and cytoplasmic translocation of HIPK1 are critical in TNFalpha-induced ASK1-JNK/p38 activation.

14-3-3 Proteins↗

PubMed2005

The calpain system is involved in the constitutive regulation of beta-catenin signaling functions.

Beta-catenin is a multifunctional protein serving both as a structural element in cell adhesion and as a signaling component in the Wnt pathway, regulating embryogenesis and tumorigenesis. The signaling fraction of beta-catenin is tightly controlled by the adenomatous polyposis coli-axin-glycogen synthase kinase 3beta complex, which targets it for proteasomal degradation. It has been recently shown that Ca(2+) release from internal stores results in nuclear export and calpain-mediated degradation of beta-catenin in the cytoplasm. Here we have highlighted the critical relevance of constitutive calpain pathway in the control of beta-catenin levels and functions, showing that small interference RNA knock down of endogenous calpain per se (i.e. in the absence of external stimuli) induces an increase in the free transcriptional competent pool of endogenous beta-catenin. We further characterized the role of the known calpain inhibitors, Gas2 and Calpastatin, demonstrating that they can also control levels, function, and localization of beta-catenin through endogenous calpain regulation. Finally we present Gas2 dominant negative (Gas2DN) as a new tool for regulating calpain activity, providing evidence that it counteracts the described effects of both Gas2 and Calpastatin on beta-catenin and that it works via calpain independently of the classical glycogen synthase kinase 3beta and proteasome pathway. Moreover, we provide in vitro biochemical evidence showing that Gas2DN can increase the activity of calpain and that in vivo it can induce degradation of stabilized/mutated beta-catenin. In fact, in a context where the classical proteasome pathway is impaired, as in colon cancer cells, Gas2DN biological effects accounted for a significant reduction in proliferation and anchorage-independent growth of colon cancer.

Adenomatous Polyposis Coli Protein↗

PubMed2005

PKCdelta mediates testosterone-induced increases in coronary smooth muscle Cav1.2.

Sex hormones have emerged as important modulators of cardiovascular physiology and pathophysiology. Our previous studies demonstrated that testosterone increases expression and activity of L-type, voltage-gated calcium channels (Cav1.2) in coronary arteries of males. The purpose of the present study was to determine whether testosterone (T) alters coronary protein kinase C delta (PKCdelta) expression and whether PKCdelta plays a role in coronary Cav1.2 expression. For in vitro studies, porcine right coronary arteries (RCA) and post-confluent (passages 3-6) 5-day, serum-restricted coronary smooth muscle cell cultures (CSMC) were incubated in the presence and absence of T or dihydrotestosterone (10 and 100 nm) for 18 h at 37 degrees C in a humidified chamber. For sex and endogenous testosterone-dependent effects, RCA were obtained from intact males, castrated males, castrated males with T replacement, and intact females. In vitro T and dihydrotestosterone caused an approximately 2-3-fold increase in PKCdelta protein levels, approximately 1.5-2-fold increase in PKCdelta kinase activity, and localization of PKCdelta toward the plasma membrane and nuclear envelope. PKCdelta protein levels were higher in coronary arteries of intact males compared with intact females. Elimination of endogenous testosterone by castration reduced RCA PKCdelta protein levels, an effect partially (approximately 45%) reversed by exogenous T (castrated males with T replacement). In CSMC, PKC inhibition with either the general PKC inhibitor, cheylerythrine, or the putative PKCdelta inhibitor, rottlerin, completely inhibited the T-mediated increase in coronary Cav1.2 protein levels. Conversely, Go6976, a conventional PKC isoform inhibitor, failed to inhibit T-induced increases in coronary Cav1.2 protein levels. PKCdelta short interference RNA completely blocked T-induced increases in Cav1.2 protein levels in CSMC. These results demonstrate for the first time that 1) endogenous T is a primary modulator of coronary PKCdelta protein and activity in males and 2) T increases Cav1.2 protein expression in a PKCdelta-dependent manner.

Animals↗

PubMed2003

Establishment of conditional vectors for hairpin siRNA knockdowns.

Small interference RNA (siRNA) is an emerging methodology in reverse genetics. Here we report the development of a new tetracycline-inducible vector-based siRNA system, which uses a tetracycline-responsive derivative of the U6 promoter and the tetracycline repressor for conditional in vivo transcription of short hairpin RNA. This method prevents potential lethality immediately after transfection of a vector when the targeted gene is indispensable, or the phenotype of the knockdown is lethal or results in a growth abnormality. We show that the controlled knockdown of DNA methyltransferase 1 (DNMT1) in human cancer resulted in growth arrest. Removal of the inducer, doxycycline, from treated cells led to re-expression of the targeted gene. Thus the method allows for a highly controlled approach to gene knockdown.

Base Sequence↗

PubMed2004

Chaperone proteins involved in troglitazone-induced toxicity in human hepatoma cell lines.

Troglitazone (TRO), an effective thiazolidinedione antidiabetic agent, was reported to produce idiosyncratic hepatotoxic effects in some individuals. In contrast, rosiglitazone (RSG), in the same group of agents, has no significant toxic effects and now is widely used. In this study, human hepatoma (HepG2) cell lines were exposed to various doses of TRO as well as RSG (0, 25, 50, and 75 microM) for 48 h. Cell lysates were separated by two-dimensional electrophoresis, and the gels were stained with coomassie brilliant blue to compare the spot profiles. The greatest protein expression at a MW of 75 kDa and isoelectric point of 5 was specifically increased with TRO treatments of 50 and 75 microM. The spot was identified as a mixture of immunoglobulin heavy chain binding protein (BiP) and, to a lesser extent, protein disulfide isomerase-related protein (PDIrp). Immunoblot analyses showed that the BiP protein was dose-dependently increased by TRO treatment and, to a lower degree, by RSG. These effects were also correlated with the high induction of BiP mRNA by TRO (50 and 75 microM) and the lower induction by RSG. However, both treatments showed no significant effects on PDIrp expression. The toxic effects of TRO in relation to the overexpression of BiP were also demonstrated in HLE cells, another human hepatoma cell line. In HLE cells, the inhibition of BiP expression by small interference RNA rendered cells more susceptible to the toxic effects of TRO. These results suggest that the overexpression of BiP is a defense mechanism of the endoplasmic reticulum in response to TRO-induced toxicity.

Amino Acid Sequence↗

PubMed2005

Ionizing radiation-induced adenovirus infection is mediated by Dynamin 2.

Specific viral targeting into intrahepatic tumors remains critical for adenovirus gene therapy in liver cancer. We previously showed that ionizing radiation increases adenovirus uptake and transgene expression in cells and colon cancer xenografts. Here, we tested whether radiation induces viral uptake through virus-cell membrane interaction. We found that radiation (8 Gy) induced adenoviral gene transfer in rat hepatocytes (WB) and human colon carcinoma cells (LoVo). This induction (24.4- and 6.5-fold, respectively) and viral uptake were significantly diminished by preincubation with antibody for Dynamin 2 but not for Coxsackie adenovirus receptor or for integrin alpha(v). Radiation-induced Dynamin 2 expression was detected by immunohistochemical staining and by increased mRNA levels for Dynamin 2 in WB (1.5-fold) and LoVo (2.2-fold) cells. Specific small interference RNA (siRNA) transfection significantly inhibited Dynamin 2 expression in various tumor cell lines (LoVo, D54, and MCF-7) and abolished the radiation induction of Dynamin 2. Likewise, radiation-induced viral gene transfer in these cells (6.5-, 5.5-, and 9.0-fold, respectively) was significantly reduced in siRNA-transfected cells (2.7-, 3.7-, and 5.0-fold, respectively). Moreover, viral uptake in LoVo tumor xenografts was significantly increased in s.c. tumors (10.9-fold) when adenovirus was given i.v. at 24 hours after tumor irradiation, coincident with an elevated Dynamin 2 expression in irradiated tumors. These data suggest that ionizing radiation induces adenovirus gene transfer in cells and tumor xenografts by regulating viral uptake, potentially through interaction with cellular Dynamin 2 and thus should provide insight into improving adenovirus targeting in tumors.

Adenoviridae↗

PubMed2006

Hypoxia-induced mitogenic factor promotes vascular adhesion molecule-1 expression via the PI-3K/Akt-NF-kappaB signaling pathway.

Hypoxia-induced mitogenic factor (HIMF), also known as FIZZ1 (found in inflammatory zone 1), is an important player in lung inflammation. However, the effects of HIMF on cell adhesion molecules involved in lung inflammation remain largely unknown. In the present work, we tested whether HIMF modulates vascular adhesion molecule (VCAM)-1 expression, and dissected the possible signaling pathways that link HIMF to VCAM-1 upregulation. Recombinant HIMF protein, instilled intratracheally into adult mouse lungs, results in a significant increase of VCAM-1 production in vascular endothelial, alveolar type II, and airway epithelial cells. In cultured mouse endothelial SVEC 4-10 and lung epithelial MLE-12 cells, we demonstrated that HIMF induces VCAM-1 expression via the phosphatidylinositol-3 kinase (PI-3K)/Akt-nuclear factor (NF)-kappaB signaling pathway. Knockdown of HIMF expression by small interference RNA attenuated LPS-induced VCAM-1 expression in vitro. We showed that HIMF induced phosphorylation of the IkappaB kinase signalsome and, subsequently, IkappaBalpha, leading to activation of NF-kappaB. Meanwhile, VCAM-1 production was correspondingly upregulated. Blocking NF-kappaB signaling pathway by expression of dominant-negative mutants of IkappaB kinase and IkappaBalpha suppressed HIMF-induced VCAM-1 upregulation. HIMF also strongly induced phosphorylation of Akt. A dominant-negative mutant of PI-3K, Deltap85, as well as PI-3K inhibitor, LY294002, also blocked HIMF-induced NF-kappaB activation and attenuated VCAM-1 production. Furthermore, LY294002 pretreatment abolished HIMF-enhanced mononuclear cells adhesion to endothelial and epithelial cells. Our findings connect HIMF to signaling pathways that regulate inflammation, and thus reveal the critical roles that HIMF plays in lung inflammation.

Animals↗

PubMed2005

In vivo gene silencing (with siRNA) of pulmonary expression of MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury.

Lung injury in trauma patients exposed to a secondary infectious/septic challenge contributes to the high morbidity/mortality observed in this population. Associated pathology involves a dys-regulation of immune function, specifically, sequestration of activated polymorphonuclear neutrophils (PMN) in the lungs. The targeting of PMN is thought to involve the release of chemokines from cells within the local environment, creating a concentration gradient along which PMN migrate to the focus of inflammation. Keratinocyte-derived chemokine (KC) and macrophage-inflammatory protein-2 (MIP-2) are murine neutrophil chemokines identified as playing significant but potentially divergent roles in the pathogenesis of acute lung injury (ALI). In the current study, we examined the contribution of local pulmonary cells to the production of KC and MIP-2 and the pathogenesis of ALI. We hypothesized that local silencing of KC or MIP-2, via the local administration of small interference RNA (siRNA) against KC or MIP-2, following traumatic shock/hemorrhage (Hem), would suppress signaling for PMN influx to the lung, thereby reducing ALI associated with a secondary septic challenge (cecal ligation and puncture). Assessment of siRNA local gene silencing was done in green fluorescent protein (GFP)-transgenic, overexpressing mice. A marked suppression of GFP expression was observed in the lung 24 h following intratracheal (i.t.) instillation of GFP siRNA, which was not observed in the liver. To test our hypothesis, siRNA against KC or MIP-2 (75 ug/C3H/Hen mouse) was instilled (i.t.) 2 h post-Hem (35 mm Hg for 90 min, 4x LRS Rx.). Twenty-four hours after, mice were subjected to septic challenge and then killed 24 h later. i.t. MIP-2 siRNA significantly (P < 0.05, ANOVA-Tukey's test, n = 5-6/group) reduced tissue and plasma interleukin (IL)-6, tissue MIP-2 (enzyme-linked immunosorbent assay), as well as neutrophil influx [myeloperoxidase (MPO) activity]. In contrast, KC siRNA treatment reduced plasma KC, tissue KC, and IL-6 but produced no significant reduction in plasma IL-6 or MPO. Neither treatment reduced tissue or plasma levels of tumor necrosis factor alpha compared with vehicle. These data support not only our hypothesis that local pulmonary chemokine production of MIP-2, to a greater extent than KC, contributes to the pathogenesis of PMN-associated ALI following Hem but also the use of siRNA as a potential therapeutic.

Animals↗

PubMed2004

Effective suppression of fibronectin synthesis by retrovirus delivered shRNA in rat cardiac fibroblasts.

Extracellular matrix overexpression is a common final pathway that leads to ventricular remodeling. Fibronectin plays a pivotal role in this progress. In the work presented here, we explored the possibility of direct inhibition of fibronectin synthesis in rat cardiac fibroblasts by small interference RNA (siRNA). We found that siRNA could successfully suppress the fibronectin overexpression stimulated by angiotensin II. To overcome the limitations of plasmid-based siRNA, we subcloned the H1 promoter into pLXIN, a commercially available retroviral vector. We found that H1 promoter worked very well to form the small hairpin RNA (shRNA) on the retroviral vector, and the fibronectin expression was dramatically down regulated by shRNA. We think the retroviral shRNA delivery system that we have constructed may have potential roles in treating ventricular remodeling.

Animals↗

PubMed2006

Identification of effective siRNA blocking the expression of SARS viral envelope E and RDRP genes.

A cell-based assay was developed to screen small interference RNA (siRNA) to block the expression of two genes of the severe acute respiratory syndrome (SARS) virus. These two genes encode RNA-dependent RNA polymerase (RDRP) and envelope E protein. The RDRP plays an essential role in viral RNA replication where envelope E protein is involved in envelope formation and virus assembly. The RDRP and envelope E genes, based on published sequences, have been synthesized and cloned into mammalian expression vectors. In addition, four siRNA sites for the RDRP gene and two siRNA sites for envelope E gene were designed and tested. The siRNA or short hairpin RNA (shRNA) expression cassettes were co-transfected with the SARS viral RDRP or envelope E expression vectors into NIH 3T3 cells. The expression levels of RDRP and envelope E genes were examined by reverse transcription followed by quantitative real-time polymerase chain reaction (PCR). Two of the siRNA expression cassettes for RDRP successfully inhibited the expression of the gene, whereas both of the siRNA expression cassettes for envelope E decreased approx 90% of the envelope E gene expression. The siRNA and shRNA for one of the siRNA sites of the RDRP gene were also tested, and it was found that both inhibited exogenous RDRP expression in a dose-dependent manner. These siRNA molecules could be used to examine the function of these genes in SARS virus replication and assembly. Furthermore, these molecules could potentially be developed into therapeutic agents for the treatment of patients with SARS.

Animals↗

PubMed2003

Histone deacetylase inhibitor up-regulates RECK to inhibit MMP-2 activation and cancer cell invasion.

Histone deacetylase (HDAC) inhibitors are known to exert antimetastatic and antiangiogenic activity in vitro and in vivo. RECK is a membrane-anchored glycoprotein that negatively regulates matrix metalloproteinases (MMPs) and inhibits tumor metastasis and angiogenesis. In this study, we test the possibility that HDAC inhibitor may increase RECK expression to inhibit MMP activation and cancer cell invasion. Our results showed that trichostatin A (TSA) up-regulated RECK via transcriptional activation in CL-1 human lung cancer cells. Flow cytometric analysis demonstrated that RECK protein on cell surface was increased after treatment of TSA. Moreover, up-regulation of RECK expression by TSA attenuated MMP-2 activity. To explore whether HDAC inhibitor-induced inhibition of MMP-2 activation is indeed mediated via RECK, we used small interference RNA (siRNA) to block RECK expression and found that inhibition of RECK by siRNA abolished the inhibitory effect of TSA on MMP-2 activation. In addition, TSA suppressed the invasive ability of CL-1 cells. Taken together, this study reveals a novel mechanism by which HDAC inhibitors suppress tumor invasion and provides a new strategy for cancer therapy.

Enzyme Activation↗

PubMed2006

[Functional study of mdr1 and GSTpi expression reversed by hairpin siRNA in K562/A02 cell line].

OBJECTIVE: To investigate the effect of hairpin small interference RNA (shRNA) on mdr1 and GSTpi protein expression in multidrug resistance human leukemia cell line K562/A02. METHOD: The shRNAs were synthesized targeting the coding region sequences of mdr1 (79 - 99 nt) and GSTpi (308 - 327 nt) respectively, and cloned to plasmid pSilencer2.1-U6 neo. The cloned products pSilence mdr1 and pSilence GSTpi were transfected into K562/A02 cells. Western blot and immunofluorescence analysis were used to detect the effectiveness and the specificity of the gene silence. 50% inhibition concentration (IC(50)) of doxorubicin (ADM) on K562/A02 cells was determined by MTT method. RESULT: pSilence mdr1 and pSilence GSTpi reduced the expression of P-gp and GSTpi protein from 0.75 +/- 0.02 and 0.54 +/- 0.02 to 0.48 +/- 0.05 and 0.39 +/- 0.02 (P < 0.01) respectively, with no effect on alpha-tubulin expression in comparison with the mock treatment. Transfection of pSilence lamin A/C into K562/A02 decreased lamin A/C expression but had no effect on the expression of P-gp and GSTpi. Immunofluorescence assay also showed that shRNAs significantly reduced the P-gp and GSTpi positive cells from (71.25 +/- 9.65)% and (81.25 +/- 6.49)% to (35.25 +/- 5.97)% and (41.25 +/- 4.43)% (P < 0.01), respectively, compared with the mock treatment. The resistance indexes after transfection were decreased to 8 (pSilence mdr1) and 10 (pSilence GSTpi) respectively from 23 (mock transfection) (P < 0.01). CONCLUSION: The shRNA could effectively and specifically reverse the multidrug resistance on K562/A02 cell line.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

[Increased sensitivity to chemotherapeutic drug by RNAi targeting Hsp701A in HepG2 cells].

AIM: To investigate the effects on chemotherapeutic sensitivity to the drugs of tumor cells induced by Hsp701A targeting siRNA expression vector. METHODS: Specific small interference RNA (siRNA) targeting Hsp701A gene was generated and its eukaryotic expression vector was constructed and transfected into human HepG2 cells.Sensitivity to the chemotherapeutic drug of HepG2 cells transfected with the vector was detected by MTT colorimetry. RESULTS: The eukaryotic expression vector of siRNA targeting Hsp701A was successfully constructed and transfected into human HepG2 cells. Sensitivity to the chemotherapeutic drug of HepG2 cells was increased after Hsp701A was knock down (P<0.05). CONCLUSION: Knockdown of Hsp701A gene by siRNA enhances sensitivity to chemotherapeutic drug in HepG2 cells.

Animals↗

PubMed2005

RNA interference-mediated virus clearance from cells both acutely and chronically infected with the prototypic arenavirus lymphocytic choriomeningitis virus.

Several arenaviruses, including Lassa fever virus, cause severe, often lethal hemorrhagic fever in humans. No licensed vaccines are available in the United States, and currently there is no efficacious therapy to treat this viral infection. Therefore the importance of developing effective antiviral approaches to combat pathogenic arenaviruses is clear. Moreover, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model for the study of viral persistence and associated diseases, as well as for exploring therapies to treat viral chronic infections. The use of small interfering RNAs (siRNAs) to downregulate gene expression via RNA interference (RNAi) has emerged as a powerful genetic tool for the study of gene function. In addition, the successful use of siRNAs to target a variety of animal viruses has led us to consider RNAi as a potential novel antiviral strategy. We have investigated the use of RNAi therapy against LCMV. Here, we show that siRNAs targeting sequences within the viral L polymerase and Z mRNAs inhibit LCMV multiplication in cultured cells. Unexpectedly, the antiviral efficacy of RNAi-based therapy against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, transfection of chemically synthesized siRNA pools to L and Z was ineffective in preventing virus multiplication. In contrast, targeting of the same viral L and Z gene products with siRNAs produced inside cells using a replication-deficient recombinant adenovirus expression system inhibited LCMV multiplication very efficiently. Notably, transduction with the replication-deficient recombinant adenovirus expression system to Z and L effectively cured persistently LCMV-infected cells, suggesting the feasibility of using RNAi therapy to combat viral chronic infections by riboviruses.

Adenoviridae↗

PubMed2005

RNA interference-mediated silencing of X11alpha and X11beta attenuates amyloid beta-protein levels via differential effects on beta-amyloid precursor protein processing.

Processing of the beta-amyloid precursor protein (APP) plays a key role in Alzheimer disease neuropathogenesis. APP is cleaved by beta- and alpha-secretase to produce APP-C99 and APP-C83, which are further cleaved by gamma-secretase to produce amyloid beta-protein (Abeta) and p3, respectively. APP adaptor proteins with phosphotyrosine-binding domains, including X11alpha (MINT1, encoded by gene APBA1) and X11beta (MINT2, encoded by gene APBA2), can bind to the conserved YENPTY motif in the APP C terminus. Overexpression of X11alpha and X11beta alters APP processing and Abeta production. Here, for the first time, we have described the effects of RNA interference (RNAi) silencing of X11alpha and X11beta expression on APP processing and Abeta production. RNAi silencing of APBA1 in H4 human neuroglioma cells stably transfected to express either full-length APP or APP-C99 increased APP C-terminal fragment levels and lowered Abeta levels in both cell lines by inhibiting gamma-secretase cleavage of APP. RNAi silencing of APBA2 also lowered Abeta levels, but apparently not via attenuation of gamma-secretase cleavage of APP. The notion of attenuating gamma-secretase cleavage of APP via the APP adaptor protein X11alpha is particularly attractive with regard to therapeutic potential given that side effects of gamma-secretase inhibition due to impaired proteolysis of other gamma-secretase substrates, e.g. Notch, might be avoided.

Adaptor Proteins, Signal Transducing↗