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PubMed2006

RNA interference-mediated prevention and therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death and is on the increase worldwide. Hepatocellular carcinoma results from chronic liver disease and cirrhosis most commonly associated with chronic hepatitis B (HBV) or hepatitis C (HCV) infection. The highest incidences of HCC are found in China and Africa, where chronic HBV infection is the major risk component. In the United States, Europe and Japan, the significant increase in HCC and HCC-related deaths within the last three decades is mainly attributed to the rise in the number of HCV-infected individuals; smaller increases of HCC are associated with HBV. Given that HCV and HBV infection account for the majority of HCCs, therapeutic and prophylactic approaches to control or eliminate virus infection may prove effective in reducing the occurrence of HCC. Although anti-viral therapies exist for both HBV and HCV infections, they are ineffective for a significant number of patients. In addition, some treatments such as interferon therapy are dose limiting owing to toxic side effects. Clearly, new approaches are needed. RNA interference (RNAi)-based approaches may meet this need and have already shown promising preclinical results in cell culture and animal models. Although this paper focuses on the potential of RNAi as a prophylactic for HCC development, the potential use of RNAi-mediated approaches for HCC therapy will also be discussed.

Animals↗
PubMed2005

Pharmacological and small interference RNA-mediated inhibition of breast cancer-associated fatty acid synthase (oncogenic antigen-519) synergistically enhances Taxol (paclitaxel)-induced cytotoxicity.

The relationship between breast cancer-associated fatty acid synthase (FAS; oncogenic antigen-519) and chemotherapy-induced cell damage has not been studied. We examined the ability of C75, a synthetic slow-binding inhibitor of FAS activity, to modulate the cytotoxic activity of the microtubule-interfering agent Taxol (paclitaxel) in SK-Br3, MDA-MB-231, MCF-7 and multidrug-resistant MDR-1 (P-Glycoprotein)-overexpressing MCF-7/AdrR breast cancer cells. When the combination of C75 with Taxol in either concurrent (C75 + Taxol 24 hr) or sequential (C75 24 hr --> Taxol 24 hr) schedules were tested for synergism, addition or antagonism using the isobologram and the median-effect plot analyses, co-exposure of C75 and Taxol mostly demonstrated synergistic effects, whereas sequential exposure to C75 followed by Taxol mainly showed additive or antagonistic interactions. Because the nature of the cytotoxic interactions was definitely schedule-dependent in MCF-7 cells, we next evaluated the effects of C75 on Taxol-induced apoptosis as well as Taxol-activated cell death and cell survival-signaling pathways in this breast cancer cell model. An ELISA for histone-associated DNA fragments demonstrated that C75 and Taxol co-exposure caused a synergistic enhancement of apoptotic cell death, whereas C75 pre-treatment did not enhance the apoptosis-inducing activity of Taxol. Co-exposure to C75 and Taxol induced a remarkable nuclear accumulation of activated p38 mitogen-activated protein kinase (p38 MAPK), which was accompanied by a synergistic nuclear accumulation of the p53 tumor-suppressor protein that was phosphorylated at Ser46, a p38 MAPK-regulated pro-apoptotic modification of p53. As single agents, FAS blocker C75 and Taxol induced a significant stimulation of the proliferation and cell survival mitogen-activated protein kinase extracellular signal-regulated kinase (ERK1/ERK2 MAPK) activity, whereas, in combination, they interfered with ERK1/ERK2 activation. Moreover, the combined treatment of C75 and Taxol inactivated the anti-apoptotic AKT (protein kinase B) kinase more than either agent alone, as evidenced by a synergistic down-regulation of AKT phosphorylation at its activating site Ser(473) without affecting AKT protein levels. To rule out a role for non-FAS C75-mediated effects, we finally used the potent and highly sequence-specific mechanism of RNA interference (RNAi) to block FAS-dependent signaling. Importantly, SK-Br3 and multi-drug resistant MCF-7/AdrR cells transiently transfected with sequence-specific double-stranded RNA oligonucleotides targeting FAS gene demonstrated hypersensitivity to Taxol-induced apoptotic cell death. Our findings establish for the first time that FAS blockade augments the cytotoxicity of anti-mitotic drug Taxol against breast cancer cells and that this chemosensitizing effect is schedule-dependent. We suggest that the alternate activation of both the pro-apoptotic p38 MAPK-p53 signaling and the cytoprotective MEK1/2 --> ERK1/2 cascade, as well as the inactivation of the anti-apoptotic AKT activity may explain, at least in part, the sequence-dependent enhancement of Taxol-induced cytotoxicity and apoptosis that follows inhibition of FAS activity in breast cancer cells. If chemically stable FAS inhibitors demonstrate systemic anticancer effects of FAS inhibition in vivo, these findings may render FAS as a valuable molecular target to enhance the efficacy of taxanes-based chemotherapy in human breast cancer.

Antineoplastic Agents, Phytogenic↗
PubMed2026

De novo transcriptome meta-analysis reveals candidate genes involved in life-stage transitions for RNAi-mediated management of the citrus root weevil (Diaprepes abbreviatus).

BACKGROUND: The citrus root weevil, Diaprepes abbreviatus, is a destructive agricultural pest for which molecular control options remain limited due to historically sparse genomic resources. Leveraging a comprehensive de novo transcriptome, we investigated developmental gene regulation across larval, pupal, and adult stages and identified essential targets for RNA interference (RNAi)-based intervention. RESULTS: Stage-resolved transcriptomic analyses revealed extensive transcriptional reprogramming associated with metabolism, detoxification, cuticle biosynthesis, endocrine signaling, and sensory perception. Among these, chitin synthase (DaCHS) emerged as a critical developmental gene, exhibiting pronounced up-regulation during late larval and pupal stages corresponding to intensive cuticle synthesis. Phylogenetic and structural analyses demonstrated that DaCHS is highly conserved among insects and retains canonical catalytic domains and transmembrane topology. Alpha Fold-based structural modeling and molecular docking confirmed stable interaction of DaCHS with its substrate, N-acetylglucosamine, supporting functional conservation of enzymatic activity. Oral delivery of DaCHS double-stranded RNA induced robust transcript suppression, leading to significant mortality and severe developmental defects, including larval and pupal abnormalities, and adults with disrupted wing and abdominal morphogenesis. CONCLUSION: These findings establish DaCHS as an indispensable gene for D. abbreviates development and validate transcriptome-guided RNAi as a powerful framework for target discovery. This work provides a strong molecular foundation for developing RNAi-based strategies that can be integrated into sustainable management programs for citrus root weevil control. © 2026 Society of Chemical Industry.

Animals↗
PubMed2005

Silencing Cenp-F weakens centromeric cohesion, prevents chromosome alignment and activates the spindle checkpoint.

Cenp-F is an unusual kinetochore protein in that it localizes to the nuclear matrix in interphase and the nuclear envelope at the G2/M transition; it is farnesylated and rapidly degraded after mitosis. We have recently shown that farnesylation of Cenp-F is required for G2/M progression, its localization to kinetochores, and its degradation. However, the role Cenp-F plays in mitosis has remained enigmatic. Here we show that, following repression of Cenp-F by RNA interference (RNAi), the processes of metaphase chromosome alignment, anaphase chromosome segregation and cytokinesis all fail. Although kinetochores attach to microtubules in Cenp-F-deficient cells, the oscillatory movements that normally occur following K-fibre formation are severely dampened. Consistently, inter-kinetochore distances are reduced. In addition, merotelic associations are observed, suggesting that whereas kinetochores can attach microtubules in the absence of Cenp-F, resolving inappropriate interactions is inhibited. Repression of Cenp-F does not appear to compromise the spindle checkpoint. Rather, the chromosome alignment defect induced by Cenp-F RNA interference is accompanied by a prolonged mitosis, indicating checkpoint activation. Indeed, the prolonged mitosis induced by Cenp-F RNAi is dependent on the spindle checkpoint kinase BubR1. Surprisingly, chromosomes in Cenp-F-deficient cells frequently show a premature loss of chromatid cohesion. Thus, in addition to regulating kinetochore-microtubule interactions, Cenp-F might be required to protect centromeric cohesion prior to anaphase commitment. Intriguingly, whereas most of the sister-less kinetochores cluster near the spindle poles, some align at the spindle equator, possibly through merotelic or lateral orientations.

Cell Cycle↗
PubMed2006

Epidermal growth factor-regulated activation of Rac GTPase enhances CD44 cleavage by metalloproteinase disintegrin ADAM10.

Invasive tumour cells, such as gliomas, frequently express EGF (epidermal growth factor) receptor at a high level and they exhibit enhanced cell migration in response to EGF. We reported previously that tumour cell migration is associated with ectodomain cleavage of CD44, the major adhesion molecule that is implicated in tumour invasion and metastasis, and that the cleavage is enhanced by ligation of CD44. In the present study, we show that EGF promotes CD44 cleavage and CD44-dependent cell migration. Introduction of a dominant-negative mutant of the small GTPase Rac1 or depletion of Rac1 by RNAi (RNA interference) abrogated CD44 cleavage induced by EGF. Treatment with PD98059, an inhibitor for MEK (mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase), also suppressed the CD44 cleavage. Furthermore, RNAi studies showed that EGF induced ADAM10 (a disintegrin and metalloproteinase 10)-dependent CD44 cleavage and cell migration. These results indicate that EGF induces ADAM10-mediated CD44 cleavage through Rac1 and mitogen-activated protein kinase activation, and thereby promotes tumour cell migration and invasion.

ADAM Proteins↗
PubMed2002

TbMP81 is required for RNA editing in Trypanosoma brucei.

Most mitochondrial mRNAs are edited in Trypano soma brucei by a series of steps that are catalyzed by a multienzyme complex that is in its initial stages of characterization. RNA interference (RNAi)-mediated repression of the expression of TbMP81, a zinc finger protein component of the complex, inhibited growth of bloodstream and insect forms, and blocked in vivo RNA editing. This repression preferentially inhibited insertion editing compared with deletion editing in vitro. It resulted in reduced specific endoribonucleolytic cleavage and a greater reduction of U addition and associated RNA ligation activities than U removal and associated RNA ligation activities. The repressed cells retained 20S editing complexes with several demonstrable proteins and adenylatable TbMP52 RNA ligase, but adenlyatable TbMP48 was not detected. Elimination of TbMP48 by RNAi repression did not inhibit cell growth or in vivo editing in either bloodstream or procyclic forms. These results indicate that TbMP81 is required for RNA editing and suggest that the editing complex is functionally partitioned.

Animals↗
PubMed2006

Criteria for effective design, construction, and gene knockdown by shRNA vectors.

BACKGROUND: RNA interference (RNAi) technology is a powerful methodology recently developed for the specific knockdown of targeted genes. RNAi is most commonly achieved either transiently by transfection of small interfering (si) RNA oligonucleotides, or stably using short hairpin (sh) RNA expressed from a DNA vector or virus. Much controversy has surrounded the development of rules for the design of effective siRNA oligonucleotides; and whether these rules apply to shRNA is not well characterized. RESULTS: To determine whether published algorithms for siRNA oligonucleotide design apply to shRNA, we constructed 27 shRNAs from 11 human genes expressed stably using retroviral vectors. We demonstrate an efficient method for preparing wild-type and mutant control shRNA vectors simultaneously using oligonucleotide hybrids. We show that sequencing through shRNA vectors can be problematic due to the intrinsic secondary structure of the hairpin, and we determine a strategy for effective sequencing by using a combination of modified BigDye chemistries and DNA relaxing agents. The efficacy of knockdown for the 27 shRNA vectors was evaluated against six published algorithms for siRNA oligonucleotide design. Our results show that none of the scoring algorithms can explain a significant percentage of variance in shRNA knockdown efficacy as assessed by linear regression analysis or ROC curve analysis. Application of a modification based on the stability of the 6 central bases of each shRNA provides fair-to-good predictions of knockdown efficacy for three of the algorithms. Analysis of an independent set of data from 38 shRNAs pooled from previous publications confirms these findings. CONCLUSION: The use of mixed oligonucleotide pairs provides a time and cost efficient method of producing wild type and mutant control shRNA vectors. The addition to sequencing reactions of a combination of mixed dITP/dGTP chemistries and DNA relaxing agents enables read through the intrinsic secondary structure of problematic shRNA vectors. Six published algorithms for siRNA oligonucleotide design that were tested in this study show little or no efficacy at predicting shRNA knockdown outcome. However, application of a modification based on the central shRNA stability should provide a useful improvement to the design of effective shRNA vectors.

Algorithms↗
PubMed2006

Are viral-encoded microRNAs mediating latent HIV-1 infection?

The Human Immunodeficiency Virus type 1 (HIV-1), a member of the lentivirus subfamily, infects both dividing and nondividing cells and, following reverse transcription of the viral RNA genome, integrates into the host chromatin where it enters into a latent state. Many of the factors governing viral latency remain unresolved and current antiviral treatment regimens are largely ineffective at eliminating cellular reservoirs of latent virus. The recent identification of microRNA (miRNA) encoding sequences embedded in the HIV-1 genome, and the discovery of functional virus-derived miRNAs, suggests a role for RNA Interference (RNAi) in the regulation of HIV-1 gene expression. Recently, the mammalian RNAi machinery was shown to regulate gene expression epigenetically by transcriptional modulation, providing a direct link between RNAi and a mechanism for inducing latency. Interestingly, both HIV-1 Tat, and the host TAR RNA-binding protein (TRBP), bind to the transactivating response (TAR) RNA of HIV-1 and affect the function of RNAi in human cells. Specifically, TRBP, a cofactor in Tat-TAR interactions, is a vital component of Dicer-mediated dsRNA processing. These novel observations support a central role for HIV-1 and associated host factors in regulating cellular RNAi and viral gene expression through RNA directed processes. Thus, HIV-1 may have evolved mechanisms to exploit the RNAi pathway at both the transcriptional and posttranscriptional level to affect and/or maintain a latent infection.

Gene Expression Regulation, Viral↗
PubMed2006

DUF283 domain of Dicer proteins has a double-stranded RNA-binding fold.

Two RNases, Dicer and Argonaute, are at the heart of the RNA interference (RNAi) molecular machinery responsible for gene silencing. Both RNases contain multiple domains, most of which have been characterized or have functions that can be predicted based on sequence comparisons. However, Dicers of higher eukaryotes contain the domain known as DUF283 which at present has no assigned role. Using sensitive profile-profile comparisons, we detected a divergent double-stranded RNA-binding domain coinciding with the DUF283 of Dicer. This finding has potential implications regarding the mechanistic role of Dicer in RNAi.

Amino Acid Sequence↗
PubMed2006

Silencing of hepatitis A virus infection by small interfering RNAs.

Infection by hepatitis A virus (HAV) can cause acute hepatitis and, rarely, fulminant liver failure, in particular in patients chronically infected with hepatitis C virus. Based on our previous observation that small interfering RNAs (siRNAs) can silence translation and replication of the firefly luciferase-encoding HAV replicon, we now exploited this technology to demonstrate the effect of siRNAs on viral infection in Huh-7 cells. Freshly and persistently infected cells were transfected with siRNAs targeting various sites in the HAV nonstructural genes. Compared to a single application, consecutive siRNA transfections targeting multiple sequences in the viral genome resulted in a more efficient and sustained silencing effect than a single transfection. In most instances, multiple applications of a single siRNA led to the emergence of viral escape mutants with mutated target sites that rendered these genomes resistant to RNA interference (RNAi). Efficient and sustained suppression of the viral infectivity was achieved after consecutive applications of an siRNA targeting a computer-predicted hairpin structure. This siRNA holds promise as a therapeutic tool for severe courses of HAV infection. In addition, the results provide new insight into the structural bases for sequence-specific RNAi.

Cell Line, Tumor↗
PubMed2003

Inhibition of virus replication by RNA interference.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism in eukaryotes, which is believed to function as a defence against viruses and transposons. Since its discovery, RNAi has been developed into a widely used technique for generating genetic knock-outs and for studying gene function by reverse genetics. Additionally, inhibition of virus replication by means of induced RNAi has now been reported for numerous viruses, including several important human pathogens such as human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, dengue virus, poliovirus and influenza virus A. In this review, we will summarize the current data on RNAi-mediated inhibition of virus replication and discuss the possibilities for the development of RNAi-based antiviral therapeutics.

Animals↗
PubMed2005

Drosophila U6 promoter-driven short hairpin RNAs effectively induce RNA interference in Schneider 2 cells.

The effect of RNA interference (RNAi) is generally more potent in Drosophila Schneider 2 (S2) cells than in mammalian cells. In mammalian cells, PolIII promoter-based DNA vectors can be used to express small interfering RNA (siRNA) or short hairpin RNA (shRNA); however, this has not been demonstrated in cultured Drosophila cells. Here we show that shRNAs transcribed from the Drosophila U6 promoter can efficiently trigger gene silencing in S2 cells. By targeting firefly luciferase mRNA, we assessed the efficacy of the shRNAs and examined the structural requirements for highly effective shRNAs. The silencing effect was dependent on the length of the stem region and the sequence of the loop region. Furthermore, we demonstrate that the expression of the endogenous cyclin E protein can be repressed by the U6 promoter-driven shRNAs. Drosophila U6 promoter-based shRNA expression systems may permit stable gene silencing in S2 cells.

Animals↗
PubMed2006

RNAi: a novel strategy for the treatment of prion diseases.

Prion disease refers to a group of fatal transmissible neurodegenerative diseases for which no pharmacological treatment is available. The cellular prion protein (PrP(C)) is required for both prion replication and pathogenesis, and reducing PrP(C) levels has been shown to extend survival time after prion infection. RNA interference (RNAi) is a sequence-specific posttranscriptional gene silencing mechanism. In this issue of the JCI, Pfeifer et al. report that lentivector-mediated RNAi significantly reduced neuronal PrP(C) expression; effectively suppressed accumulation of the infectious protease-resistant form of PrP (PrP(Sc)) in a persistently infected neuroblastoma cell line; and markedly slowed the progression of prion disease in a unique chimeric mouse model (see the related article beginning on page 3204). These findings indicate that lentivector-mediated RNAi could, in principle, be developed for the therapy of prion disease.

Animals↗
PubMed2005

RNA interference as a gene-specific approach for molecular medicine.

The discovery of RNA interference (RNAi) in eukaryotic cells has been the major recent breakthrough in molecular and cell biology. RNAi machineries exert biological functions in gene regulation, genome defense and chromatin architecture and dynamics. The potential of RNAi to silence any gene of interest in a highly specific and efficient manner via double-stranded RNA (dsRNA) has literally revolutionized modern genetics. RNAi-based functional genomics now permits, for the first time, to evaluate the cellular role of individual gene products on a genome-wide scale in higher organisms like mammals, presenting an alternative to the generation of animal knockouts often doomed to failure because of a lethal phenotype. RNAi has had an enormous impact on the development of novel disease models in animals, and it is likely that small interfering RNAs (siRNAs), which are the trigger molecules for RNA silencing, will become an invaluable tool for the treatment of genetic diseases. First clinical trials, using siRNAs directed against the vascular endothelial growth factor (VEGF) or one of its receptors, have been initiated recently for the treatment of age-related macular degeneration. Improving guidelines for the rational design of siRNAs, based on recent progress in understanding the mechanisms underlying RNAi, as well as the introduction of chemical modifications into siRNAs are expected to improve their pharmacokinetic and pharmacodynamic properties for in vivo applications. Finally, successful therapeutic application of RNAi will depend on the development of improved siRNA delivery strategies that combine high specificity and efficiency with a low immunostimulatory and tumorigenic potential.

Chemistry, Pharmaceutical↗
PubMed2003

Transgenic RNA interference in ES cell-derived embryos recapitulates a genetic null phenotype.

Gene targeting via homologous recombination in murine embryonic stem (ES) cells has been the method of choice for deciphering mammalian gene function in vivo. Despite improvements in this technology, it still remains a laborious method. Recent advances in RNA interference (RNAi) technology have provided a rapid loss-of-function method for assessing gene function in a number of organisms. Studies in mammalian cell lines have shown that introduction of small interfering RNA (siRNA) molecules mediates effective RNA silencing. Plasmid-based systems using RNA polymerase III (RNA pol III) promoters to drive short hairpin RNA (shRNA) molecules were established to stably produce siRNA. Here we report the generation of knockdown ES cell lines with transgenic shRNA. Because of the dominant nature of the knockdown, embryonic phenotypes could be directly assessed in embryos completely derived from ES cells by the tetraploid aggregation method. Such embryos, in which endogenous p120-Ras GTPase-activating protein (RasGAP), encoded by Rasa1 (also known as RasGAP), was silenced, had the same phenotype as did the previously reported Rasa1 null mutation.

Animals↗
PubMed2006

[Effects of RNA interference targeting angiotensin 1a receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C hypertension].

OBJECTIVE: To investigate the effects of RNA interference (RNAi) targeting angiotensin 1a (AT1a) receptor on the blood pressure and cardiac hypertrophy of rats with 2K1C (2-kidney, 1-clip) hypertension. METHODS: Two kinds of RNAi plasmids, pAT1a-shRNA1 carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 928 - 946 and pAT1a-shRNA2 carrying an U6 promoter and an AT1a-specific shRNA-coding template sequence corresponding the sites 978 - 996, and a blank plasmid pCon carrying a nonspecific shRNA-coding sequence were constructed. Thirty Sprague-Dawley rats underwent clipping of the left renal artery so as to establish two-kidney, one-clip (2K1C) hypertension models and then were randomly divided into 5 equal groups: pAT1a-shRNA1 group (injected with pAT1a-shRNA1 4 mg/kg only one time), pAT1a-shRNA2 group (injected with pAT1a-shRNA2 4 mg/kg only one time), pCon group (injected with pCon 4 mg/kg only one time), valsartan group (perfused into the stomach with valsartan, a AT1 receptor inhibitor 30 mg.kg(-1).d(-1), for 3 weeks), and control blank group (without any treatment). Three weeks later, the systolic pressure of the caudal artery was measured, catheterization through carotid artery was conducted to measure the systolic blood pressure (SBP) and diastolic blood pressure (DBP), and the left ventricular pressure curve was drawn. Then the rats were killed; the weight of the heart was measured, the ratio of left ventricle weight to body weight (LV/BW) was calculated, and pathological examination of the heart and thoracic aorta was performed. Western blotting was used to detect the protein expression of AT21 in the ventricle and aorta. Six age-matched healthy rats were used as normal controls. RESULTS: There was no significant difference in the caudal artery pressure among the 5 groups (all P > 0.05) before intervention. Three weeks later the caudal artery pressures of the blank control group and pCon group continued to significantly increase by about 25 mm Hg compared to the values before the intervention (both P < 0.001) and without significant difference between these 2 groups; however, the caudal artery pressures of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 15.1 mm Hg +/- 5.4 mm Hg, 16.4 mm Hg +/- 8.4 mm Hg, and 30.6 mm Hg +/- 18.2 mm Hg lower than those before the intervention respectively (all P < 0.01); and were also significantly lower than those of the blank groups (P < 0.01 or P < 0.05). There was no significant differences in the +/- dp/dt value and indicators of renal function among these groups. The carotid artery pressure of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 194 mm Hg +/- 5 mm Hg, 200 mm Hg +/- 5 mm Hg, and 164 mm Hg +/- 5 mm Hg, all significantly lower than those of the blank and pCon groups (234 mm Hg +/- 10 mm Hg and 232 mm Hg +/- 7 mm Hg respectively, all P < 0.01). The LV/BW of the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups were 2.27 +/- 0.37, 2.31 +/- 0.26, and 2.26 +/- 0.39, all significantly lower than that of the blank and pCon groups (3.24 +/- 0.38 and 2.94 +/- 0.06, respectively, all P < 0.01), similar to that of the normal control group (P > 0.05). The myocardiocytes were significantly hypertrophic and the arterial tunica media was significantly thickened in the blank group and such changes were all improved to different degrees in the pAT1a-shRNA1, pAT1a-shRNA2, and valsartan groups. The protein expression levels of AT1 receptor in the myocardium of the pAT1a-shRNA and pAT1a-shRNA2 groups were lower by 53.3% and 47.8% respectively than that of the blank group, and the protein expression levels of AT1 receptor in the thoracic aorta of the pAT1a-shRNA and pAT1a-shRNA2 groups were lower by 58.7% and 49.3% respectively than that of the blank group (all P < 0.01); however, there were no significant difference in the protein expression levels of AT1 receptor in the myocardium and thoracic aorta between the valsartan and blank groups (both P > 0.05). CONCLUSION: RNA interference targeting AT1a receptor inhibits the development of renovascular hypertension and the accompanying cardiac hypertrophy. The RNAi technology may become a new strategy of gene therapy for hypertension.

Animals↗
PubMed2005

Molecular consequences of silencing mutant K-ras in pancreatic cancer cells: justification for K-ras-directed therapy.

Mutation of the K-ras gene is an early event in the development of pancreatic adenocarcinoma and, therefore, RNA interference (RNAi) directed toward mutant K-ras could represent a novel therapy. In this study, we examine the phenotypic and molecular consequences of exposure of pancreatic tumor cells to mutant-specific K-ras small interfering RNA. Specific reduction of activated K-ras via RNAi in Panc-1 and MiaPaca-2 cells resulted in cellular changes consistent with a reduced capacity to form malignant tumors. These changes occur through distinct mechanisms but likely reflect an addiction of each cell line to oncogene stimulation. Both cell lines show reduced proliferation after K-ras RNAi, but only MiaPaca-2 cells showed increased apoptosis. Both cell lines showed reduced migration after K-ras knockdown, but changes in integrin levels were not consistent between the cell lines. Both cell lines showed alteration of the level of GLUT-1, a metabolism-associated gene that is downstream of c-myc, with Panc-1 cells demonstrating decreased GLUT-1 levels, whereas MiaPaca-2 cells showed increased levels of expression after K-ras knockdown. Furthermore, after K-ras RNAi, there was a reduction in angiogenic potential of both Panc-1 and MiaPaca-2 cells. Panc-1 cells increased the level of expression of thrombospondin-1, an endogenous inhibitor of angiogenesis, whereas MiaPaca-2 cells decreased the production of vascular endothelial growth factor, a primary stimulant of angiogenesis in pancreatic tumors. We have found that silencing mutant K-ras through RNAi results in alteration of tumor cell behavior in vitro and suggests that targeting mutant K-ras specifically might be effective against pancreatic cancer in vivo.

Adenocarcinoma↗
PubMed2006

[Inhibition of HLA-DRB1*0405 gene expression by siRNA].

OBJECTIVE: To investigate the effects of small interfering RNA (siRNA) of HLA DRB1(*)0405 HLA-DRB1(*)0405 gene expression with plasmid-based siRNAs. METHOD: Plasmid expressing HLA-DRB1(*)0405-renilla fusion protein-siCHECK-2/HLA-DRB1(*)0405, 6 different short hairpin RNAs (shRNAs) targeting 6 19 bp nucleotide sequences of HLA-DRB1(*)0405 (siRNA1 approximately 6), and one shRNA targeting the control non-specific sequence (siRNAC) were designed and constructed. Human embryonic kidney cells of the line 293 were cultured and co-transfected by lipids some with the plasmid siCHECK-2/HLA-DRB1(*)0405 and one specific shRNA expressing vector transiently, and cells without shRNA-transfection were used as negative controls. The impact of RNAi on HLA-DRB1(*)0405 expression was analyzed by real time fluorescence quantification RT-PCR and luciferase test. RESULTS: The expression of HLA-DRB1(*)0405 gene RNA of the 293 cells transfected with siRNA1, siRNA2, siRNA3, siRNA, and siRNA6 were down-regulated to 10.75%, 83.22%, 30.63%, 48.54%, and 89.92% that of the control group with the inhibition rates of 89.25%, 16.78%, 69.37%, 51.46%, and 10.08% respectively. However, no significant downregulation was showed in the cells transfected with siRNA4 and siRNAC. The 293 cells transfected with siRNA1 and siRNA 3 showed a significant downregulation of the protein expression of HLA-DRB1(*)0405 gene with the inhibitory rates of 6.70% and 36.85% respectively; however, the cells transfected with siRNA2, siRNA4, siRNA5, and siRNA6 did not show a significant downregulation. CONCLUSION: The significant inhibition of HLA-DRB1(*)0405 gene expression by siRNA suggests a therapeutic approach in rheumatoid arthritis: to use RNA interference (RNAi) to inhibit the abnormal immune reaction mediated by HLA-DRB1 in rheumatoid arthritis.

Arthritis, Rheumatoid↗