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PubMed2003

RNA interference: gene silencing in the fast lane.

Sequencing of whole genomes has provided new perspectives into the blueprints of diverse organisms. Knowing the sequences, however, does not always tell us much about the function of the genes that regulate development and homeostasis. RNA interference (RNAi) is becoming the method of choice for gene function analysis in cells and whole organisms. Here we review the approaches available to perform RNAi experiments in mammalian cells and in mice. We discuss usage of RNAi in cancer research and as a possible therapeutic tool for cancer treatment.

Animals↗

PubMed2006

RNA interference of the salivary gland nitrophorin 2 in the triatomine bug Rhodnius prolixus (Hemiptera: Reduviidae) by dsRNA ingestion or injection.

Mass sequencing of cDNA libraries from salivary glands of triatomines has resulted in the identification of many novel genes of unknown function. The aim of the present work was to develop a functional RNA interference (RNAi) technique for Rhodnius prolixus, which could be widely used for functional genomics studies in triatomine bugs. To this end, we investigated whether double-stranded RNA (dsRNA) can inhibit gene expression of R. prolixus salivary nitrophorin 2 (NP2) and what impact this might have on anticoagulant and apyrase activity in the saliva. dsRNA was introduced by two injections or by ingestion. RT-PCR of the salivary glands showed that injections of 15 microg of NP2 dsRNA in fourth-instar nymphs reduced gene expression by 75+/-14% and that feeding 1 microg/microL of NP2 dsRNA into second-instar nymphs (approx. 13 microg in total) reduced gene expression by 42+/-10%. Phenotype analysis showed that saliva of normal bugs prolonged plasma coagulation by about four-fold when compared to saliva of knockdown bugs. These results and the light color of the salivary gland content from some insects are consistent with the knockdown findings. The findings suggest that RNAi will prove a highly valuable functional genomics technique in triatomine bugs. The finding that feeding dsRNA can induce knockdown is novel for insects.

Animals↗

PubMed2006

[Effect of silencing epidermal growth factor receptor expression by RNA interference on the growth of nasopharyngeal carcinoma cell 5-8F].

OBJECTIVE: To observe the growth and cell cycle changes of nasopharyngeal carcinoma cell 5-8F in response to silencing of the expression of epidermal growth factor receptor (EGFR) with RNA interference (RNAi), and explore the possible relationships between EGFR and the occurrence, differentiation and progression of nasopharyngeal carcinoma. METHOD: Three EGFR-specific small interfering RNAs (siRNAs) were obtained by in vitro transcription and synthesis and were transiently transfected into 5-8F cells. The EGFR expression levels in the transfected cells were detected by reverse transcription (RT)-PCR and Western blotting, respectively. After EGFR expression silencing, the growth and cell cycle changes of the cells were observed. RESULTS: EGFR mRNA contents and protein levels decreased by approximately 67.5% and 77%, respectively, after RNAi of 5-8F cells, and the cell proliferation decreased and cell cycle arrest at G1 phase occurred in association with EGFR silencing. CONCLUSION: EGFR silencing by RNAi can reduce the proliferation of nasopharyngeal carcinoma cells and induce cell cycle arrest at G1 phase, which sheds light on the possible use of RNAi for further investigation of the pathogenesis and gene therapy of nasopharyngeal carcinoma.

Cell Line, Tumor↗

PubMed2003

Using RNA interference to manipulate endogenous gene expression in Schistosoma mansoni sporocysts.

Direct assessments of gene function in parasitic flatworms have been hampered by the lack of effective tools to alter gene expression. The aim of the present study was to use RNA-interference (RNAi) to achieve targeted gene knockdown in larval stages of the human blood fluke, Schistosoma mansoni. We selected two S. mansoni genes for RNAi experiments: SGTP1, a facilitated diffusion glucose transporter and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). When S. mansoni larvae were treated in vitro for 6 days with dsRNA specific to one of these two genes, targeted transcript levels were reduced by 70-80% as determined by quantitative PCR (qPCR), while non-targeted transcripts were unaffected. Parasite exposure to SGTP1 dsRNA, but not GAPDH dsRNA, reduced larval glucose-uptake capacity by 40%, demonstrating that SGTP1 transcript knockdown results in the functional phenotype of reduced glucose transport activity. The effect of dsRNA treatment on transcript level was evident for up to 28 days after an initial dsRNA treatment. Interestingly, dsRNA treatment was effective only when miracidia were allowed to undergo the transition to sporocysts in its presence, while treatment of fully transformed sporocysts was ineffective. Fluorescence patterns in larvae exposed to rhodamine-labeled dsRNA as miracidia and sporocysts were similar, suggesting that the difference in susceptibility to dsRNA treatment between the two life stages may not be due to differences in dsRNA entry. Overall, this technology will enable direct assessment of the roles of individual genes in physiological processes of larval stages of S. mansoni, a crucial step in the identification of novel intervention targets for this important human pathogen.

Animals↗

PubMed2006

Reinstalling antitumor immunity by inhibiting tumor-derived immunosuppressive molecule IDO through RNA interference.

Tumor-derived immune suppression is a major impediment to successful immune/gene cancer therapy. In the present study, we describe a novel strategy to disrupt tumor-derived immune suppression by silencing a tolerogenic molecule of tumor origin, IDO, using small interfering RNA (siRNA). Silencing of IDO in B16F10 cells in vitro using IDO-siRNA prevented catabolism of tryptophan and inhibited apoptosis of T cells. IDO-siRNA treatment of B16F10 cells in vitro inhibited subsequent growth, tumor formation, and the size of tumor formed, by those cells when transplanted into host mice. In vivo treatment of B16F10 tumor-bearing mice successfully postponed tumor formation time and significantly decreased tumor size. Furthermore, in vivo IDO-siRNA treatment resulted in recovery of T cells responses and enhancement of tumor-specific killing. Thus, silencing IDO may break tumor-derived immune suppression. These data indicate that RNA interference has potential to enhance cancer therapy by reinstalling anticancer immunity.

Animals↗

PubMed2005

[c-myc gene silencing in K562 cells with RNA interference].

OBJECTIVE: To study the inhibitory effect of small interfering RNA (siRNA) targeting c-myc gene in K562 cells. METHODS: siRNAs targeting the site 1357 of c-myc mRNA was designed and synthesized. In vitro cultured K562 cells were transfected with lipofectamine 2000 and the inhibitory effect was detected by reverse transcriptase (RT)-PCR, cell count, MTT assay and fluorescence-activated cell sorting. RESULTS: Compared with the negative and blank control group, the transfection group showed marked decrease in the c-myc expression and the K562 cells exhibited increased apoptosis rate. CONCLUSION: RNA interference can effectively inhibit c-myc expression and induce apoptosis in K562 cells.

Apoptosis↗

PubMed2004

The RNA interference proteins and vasa locus are involved in the silencing of retrotransposons in the female germline of Drosophila melanogaster.

RNA interference (RNAi) is considered as a defense against expansion of transposable elements. The proteins related to RNA helicase and Argonaute families are involved in RNAi process in different organisms. It was shown that Argonaute AUBERGINE and putative RNA helicase SPINDLE-E proteins were essential for RNAi in Drosophila. Here, we describe the role of aubergine (aub) and spindle-E (spn-E) genes in the control of LTR retrotransposon copia and nonLTR telomeric Het-A and I retrotransposons in ovaries. spn-E mutation causes a drastically increased lacZ expression driven by copia LTR. For the first time we show the involvement of AUBERGINE protein and VASA RNA helicase, essential for oocyte patterning, in the retrotransposon silencing. spn-E, vasa and aub mutations cause similar accumulation of both I element and Het-A transcripts in the developing oocyte. VASA and AUBERGINE proteins are known as components of perinuclear ribonucleoprotein particles in germ cells, and spn-E mutation disturbs protein content of the particles. We suggest participation of these proteins in the same silencing pathway.

Animals↗

PubMed2005

Stable suppression of tumorigenicity by Pin1-targeted RNA interference in prostate cancer.

PURPOSE: The peptidyl-prolyl isomrase Pin1 plays a catalytic role in oncogenesis in solid cancers, including prostate cancer. In the present study, we sought to determine the potential of Pin1-targeted gene silencing in inhibiting cellular growth and tumorigenicity in prostate cancer. EXPERIMENTAL DESIGN: A retrovirus-mediated RNA interference targeting Pin1 was expressed in PC3 and LNCaP cells, and cell growth and several transformed properties were investigated. RESULTS: The stable expression of Pin1-specific small interfering RNA constructs in PC3 and LNCaP cells significantly reduced cellular proliferation, colony formation, migration, and invasion but strongly enhanced the apoptotic response induced by serum depletion or treatment with anticancer agents. Furthermore, Pin1 depletion significantly suppressed tumorigenic potential in athymic mice, resulting in the inhibition of both tumor growth and angiogeneisis. CONCLUSIONS: These results strongly suggest that Pin1 plays an important role not only in tumorigenesis but also in the maintenance of the transformed phenotype in prostate cancer cells. Hence, Pin1 may serve as a promising therapeutic target, particularly for recurrent prostate tumors.

Animals↗

PubMed2006

XIAP is related to the chemoresistance and inhibited its expression by RNA interference sensitize pancreatic carcinoma cells to chemotherapeutics.

OBJECTIVES: To explore the exact role does the x-linked inhibitor of apoptosis (XIAP) play in chemoresistance of pancreatic carcinoma cell and the cell sensitivity to chemotherapeutic drugs changed after XIAP is inhibited by RNA interference (RNAi). METHODS: Pancreatic carcinoma cell line SW1990 was exposed to 5-fluorouracil (5-fu) with the concentrations of 1.0 and 10 mug/mL to increase the expression of XIAP. Then 4 RNAi plasmid vectors against XIAP were designed and constructed, then transfected into SW1990. Repressive effect was evaluated by reverse transcriptase polymerase chain reaction and Western blot; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry were performed to determine cell sensitivity to 5-fu and gemcitabine; furthermore, apoptosis is confirmed by Hoechst 33258 stain. RESULTS: XIAP of SW1990 can be up-regulated with the chemoresistance increasing 1.5- and 4-fold after 10 and 30 days induced by 5-fu. Two of the 4 vectors can inhibit the expression of XIAP protein more than 60%. The cells apoptosis index induced by 5-fu and gemcitabine increased greatly after XIAP is inhibited by the RNAi plasmid vectors. CONCLUSIONS: XIAP is one of the most important factors in the pancreatic carcinoma chemoresistance, and inhibition of XIAP in pancreatic carcinoma can enhance the cancer sensitivity to chemotherapeutic drugs.

Apoptosis↗

PubMed2005

Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS.

Mutations in Cu/Zn superoxide dismutase (encoded by SOD1), one of the causes of familial amyotrophic lateral sclerosis (ALS), lead to progressive death of motoneurons through a gain-of-function mechanism. RNA interference (RNAi) mediated by viral vectors allows for long-term reduction in gene expression and represents an attractive therapeutic approach for genetic diseases characterized by acquired toxic properties. We report that in SOD1(G93A) transgenic mice, a model for familial ALS, intraspinal injection of a lentiviral vector that produces RNAi-mediated silencing of SOD1 substantially retards both the onset and the progression rate of the disease.

Amyotrophic Lateral Sclerosis↗

PubMed2026

Exploring the potential of RNA interference (RNAi) in mosquito control: from mechanisms to molecular insights.

Mosquito-borne diseases represent a growing global health crisis, exacerbated by climate change and insecticide resistance. RNA interference (RNAi), a natural mechanism of gene silencing, offers a promising, target-specific alternative for mosquito control. This review explores the potential of RNAi to disrupt critical physiological processes, such as reproduction and disease transmission, thereby reducing vector populations and competence. We examine the mechanisms of RNAi, its application in combatting insecticide resistance, and recent advancements in delivery systems, including nanobody- and chitosan-based nanoparticles, which enhance the stability and uptake of double-stranded RNA (dsRNA) molecules. However, significant challenges remain, such as optimizing field-effective delivery methods and assessing potential off-target effects on non-target organisms. Continued innovation in RNAi technology is pivotal for developing sustainable and environmentally sound vector control strategies. This review synthesizes current research, highlighting the molecular insights, practical applications, and future directions for integrating RNAi into modern public health initiatives.

RNA Interference↗

PubMed2005

Application of RNA interference to root-knot nematode genes encoding esophageal gland proteins.

Plant parasitic nematodes have been, so far, refractory to transformation or mutagenesis. The functional analysis of nematode genes relies on the development of reverse genetic tools adapted to these obligate parasites. Here, we describe the application of RNA interference (RNAi) to the root-knot nematode Meloidogyne incognita for the knock-down of two genes expressed in the subventral esophageal glands of the nematode and potentially involved in parasitism, the calreticulin (Mi-crt) and the polygalacturonase (Mi-pg-1) genes. Incubation in 1% resorcinol for 4 h induced double-stranded RNA uptake through the alimentary track of the nematodes and led to up to 92% depletion of Mi-crt transcripts. Timecourse analysis of the silencing showed different temporal patterns for Mi-crt and Mi-pg-1. The silencing of Mi-crt was optimal 20 h after soaking, whereas the silencing of Mi-pg-1 was optimal 44 h after soaking. For the two genes, the silencing effect was highly time-limited, since no transcript depletion was detectable 68 h after soaking.

Animals↗

PubMed2004

Paradoxical actions of endogenous and exogenous insulin-like growth factor-binding protein-5 revealed by RNA interference analysis.

Insulin-like growth factor-binding protein-5 (IGFBP-5) is abundantly expressed in bone cells. To determine the physiological role(s) of endogenous IGFBP-5 in regulating bone cell growth, differentiation, and survival, we used short double-stranded RNA (siRNA) to trigger RNA interference of IGFBP-5 in human osteosarcoma cells. The IGFBP-5 siRNA, targeting against a sequence unique to the IGFBP-5 middle domain, efficiently reduced IGFBP-5 mRNA and protein levels. The IGFBP-5 siRNA did not change the levels of IGFBP-4, a structurally related protein, or glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene. Knock-down of IGFBP-5 resulted in a significant increase in the number of transferase-mediated dUTP nick end labeling-positive cells and a decrease in a bone differentiation parameter (alkaline phosphatase activity) but had little effect on basal or insulin-like growth factor I-induced proliferation. Overexpression of a siRNA-resistant IGFBP-5 mutant in the IGFBP-5 knock-down cells restored the levels of survival to the control level; overexpression of IGFBP-4 or wild type IGFBP-5 had no such effect. Paradoxically, the addition of exogenous IGFBP-5 not only failed to rescue IGFBP-5 knock-down-induced apoptosis, it caused a further increase in apoptosis. Furthermore, the addition of exogenous IGFBP-5 alone increased apoptosis. This pro-apoptotic action of exogenous IGFBP-5 was abolished when IGF-I was added in excess, suggesting that exogenous IGFBP-5 increases apoptosis by binding to and inhibiting the activities of insulin-like growth factors. These results indicate that endogenous and exogenous IGFBP-5 exhibits opposing biological actions on cell survival and underscore the necessity and utility of studying IGFBP functions through loss-of-function approaches.

Alkaline Phosphatase↗

PubMed2004

RNA interference: historical overview and significance.

In the early 1990s, attempts to manipulate gene expression by researchers working in three different fields resulted in unanticipated gene silencing. Rather than ignoring such results, these researchers went on to document and further investigate the nature of such silencing, which was named "co-suppression" in plants, "quelling" in fungi, and "RNA interference" (RNAi) in nematodes. By the late 1990s, it was discovered that silencing could be initiated in this diverse set of organisms by exposing cells to double-stranded RNA (dsRNA), which directed the destruction of mRNAs containing similar sequences. Soon afterward, such dsRNA-mediated silencing was employed as a reverse genetic technique to analyze the functions of specific genes in a broad variety of organisms. Biochemical and genetic studies designed to uncover the components of the RNA silencing machinery identified a common core of proteins that serve to amplify the interfering RNA signal and direct endonucleolytic cleavage of target RNAs. A subset of silencing events may also direct DNA methylation of targeted genes. RNA silencing is thought to have evolved as a defense mechanism to suppress viral replication and transposon mobilization. However, additional functions involving the RNAi machinery have been uncovered, including posttranscriptional regulation of endogenous genes, and maintenance of structure and function of heterochromatin. Whereas many researchers have focused on understanding the natural biological functions of RNA silencing, others are testing its utility in antiviral and cancer therapies and in other biotechnological and biomedical applications.

Animals↗

PubMed2005

RNA interference targeting aurora kinase a suppresses tumor growth and enhances the taxane chemosensitivity in human pancreatic cancer cells.

AURKA/STK15/BTAK, the gene encoding Aurora A kinase that is involved in the regulation of centrosomes and segregation of chromosomes, is frequently amplified and overexpressed in various kinds of human cancers, including pancreatic cancer. To address its possibility as a therapeutic target for pancreatic cancer, we employed the RNA interference technique to knockdown AURKA expression and analyzed its phenotypes. We found that the specific knockdown of AURKA in cultured pancreatic cancer cells strongly suppressed in vitro cell growth and in vivo tumorigenicity. The knockdown induced the accumulation of cells in the G(2)-M phase and eventual apoptosis. Furthermore, we observed a synergistic enhancement of the cytotoxicity of taxanes, a group of chemotherapeutic agents impairing G(2)-M transition, by the RNA interference-mediated knockdown of AURKA. These results indicate that inhibition of AURKA expression can result in potent antitumor activity and chemosensitizing activity to taxanes in human pancreatic cancer.

Apoptosis↗

PubMed2003

Gene silencing in Caenorhabditis elegans by transitive RNA interference.

When a cell is exposed to double-stranded RNA (dsRNA), mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi). Here, we provide evidence that dsRNA is amplified in Caenorhabditis elegans to ensure a robust RNAi response. Our data suggest a model in which mRNA targeted by RNAi functions as a template for 5' to 3' synthesis of new dsRNA (termed transitive RNAi). Strikingly, the effect is nonautonomous: dsRNA targeted to a gene expressed in one cell type can lead to transitive RNAi-mediated silencing of a second gene expressed in a distinct cell type. These data suggest dsRNA synthesized in vivo can mediate systemic RNAi.

Animals↗

PubMed2003

Relative contribution of Nedd4 and Nedd4-2 to ENaC regulation in epithelia determined by RNA interference.

Epithelial Na+ transport is regulated in large part by mechanisms that control expression of the epithelial Na+ channel (ENaC) at the cell surface. Nedd4 and Nedd4-2 are candidates to control ENaC surface expression, but it is not known which of these proteins contributes to ENaC regulation in epithelia. To address this question, we used RNA interference to selectively reduce expression of Nedd4 or Nedd4-2. We found that endogenous Nedd4-2, but not Nedd4, negatively regulates ENaC in two epithelial cell lines (Fischer rat thyroid and H441); small interfering RNA (siRNA) against Nedd4-2 increased amiloride-sensitive Na+ current (compared with control siRNA), but Nedd4 siRNA did not. A mutation associated with Liddle's syndrome (betaR566X) abolished the effect of Nedd4-2 siRNA, suggesting that a defect in ENaC regulation by Nedd4-2 contributes to the pathogenesis of this inherited form of hypertension. Previous work found that Nedd4-2 is phosphorylated by serum and glucocorticoid-regulated kinase, a Ser/Thr kinase induced by steroid hormones. Here we found that Nedd4-2 phosphorylation contributes to ENaC regulation by steroid hormones. Consistent with this model, ENaC stimulation by dexamethasone was reduced by Nedd4-2 siRNA and by overexpression of a mutant Nedd4-2 lacking serum and glucocorticoid-regulated kinase phosphorylation sites. Thus, endogenous Nedd4-2 negatively regulates ENaC in epithelia and is a component of a signaling pathway by which steroid hormones regulate ENaC. Defects in this regulation may contribute to the pathogenesis of hypertension.

Animals↗

PubMed2001

Suppression of gene expression by RNA interference in cultured plant cells.

Suppression by double-stranded RNA (dsRNA) of the expression of a target gene is known as RNA interference (RNAi). No quantitative analysis of the effects of RNAi on the expression of specific genes in cultured plant cells has been reported. However, as it is possible to produce populations of cultured plant cells that are uniform and divide synchronously for functional analysis of genes of interest, we performed a quantitative study of the effects of RNAi in such cells. We constructed dsRNA expression plasmids for a luciferase gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter by simply connecting sense and antisense sequences in a head-to-head manner. An RNAi effect was observed 24 hours after the introduction of dsRNA expression plasmids into tobacco BY-2 cells by electroporation. The simple system for suppression of specific genes in plant cells should be useful in attempts to elucidate the roles of individual genes in plant cells.

Animals↗