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Results for “Small interfering RNA”

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PubMed2003

Small interfering RNA suppression of polyamine analog-induced spermidine/spermine n1-acetyltransferase.

N1,N11-diethylnorspermine (DENSPM) is a polyamine analog that down-regulates polyamine biosynthesis and potently upregulates the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). In certain cells, such as SKMEL-28 human melanoma cells, induction of SSAT is associated with rapid apoptosis. In this study, we used small interfering RNA (siRNA) to examine the role of SSAT induction in mediating polyamine pool depletion and apoptosis. siRNA duplexes were designed to target three independent sites in the SSAT mRNA coding region (siSSAT). When transfected under nontoxic conditions, two of the duplexes selectively reduced basal SSAT mRNA in HEK-293 cells by >80% and prevented DENSPM-induced SSAT mRNA by 95% in SK-MEL-28 cells. Treatment of SK-MEL-28 cells with 10 muM DENSPM in the presence of 83 nM siSSAT selectively prevented the 1400-fold induction of SSAT activity by approximately 90% and, in turn, prevented analog depletion of spermine (Spm) pools by approximately 35%. siSSAT also prevented DENSPM-induced cytochrome c release and caspase-3 cleavage at 36 h and apoptosis at 48 h as measured by annexin V staining. Overall, the data directly link analog induction of SSAT to Spm pool depletion and to caspase-dependent apoptosis in DENSPM-treated SK-MEL-28 cells. This represents the first use of siRNA technology directed toward a polyamine gene and the first unequivocal demonstration that SSAT induction initiates events leading to polyamine analog-induced apoptosis.

Acetyltransferases↗

PubMed2004

Knockdown of survivin expression by small interfering RNA reduces the clonogenic survival of human sarcoma cell lines independently of p53.

Survivin, a member of the inhibitors-of-apoptosis gene family, is overexpressed in many tumor types. Survivin is a prognostic marker of soft-tissue sarcomas, but the downregulation of survivin expression and the possible dependency of survivin downregulation on p53 in these tumors have not been investigated. Therefore, we applied small interfering RNA (siRNA) to knock down the expression of survivin in five human sarcoma cell lines with wild-type or mutant p53 alleles. Compared with survivin mRNA expression in the nonsense siRNA-treated sarcoma cell lines, expression after treatment with survivin-specific siRNA was reduced by 73-88%; survivin protein expression was reduced by 52-81%. This finding was coupled with a reduction in clonogenic survival ranging from 65-86%. However, less than 10% of cells treated with survivin-specific siRNA underwent apoptosis. Cell-cycle and morphologic analyses showed that after a dramatic increase in the number of treated cells in the G2/M phase, some of the cells became polyploid; this result indicates that mitosis of a substantial number of treated cells was incomplete. Our findings suggest that survivin-specific siRNA could be a selective treatment to kill sarcoma cells regardless of the presence or absence of wild-type p53 alleles.

Apoptosis↗

PubMed2006

Inhibition of hypoxia-inducible factor-1alpha and endothelial progenitor cell differentiation by adenoviral transfer of small interfering RNA in vitro.

RNA interference is applied to study gene function in different organisms and in various cell types. Little is known about the effect of RNA interference on human endothelial progenitor cells (EPCs) in vitro. To address this issue, short hairpin RNA targeting the human hypoxia inducible factor-1alpha (HIF-1alpha) was transferred into human EPCs by an adenoviral vector. HIF-1alpha mRNA and protein expression was dramatically and specifically downregulated after adeno-small interfering RNA (siRNA)-HIF-1alpha infection in cells under hypoxia, a condition in which HIF-1alpha would have been induced. This effect persisted for at least 72 h and was accompanied by suppression of vascular endothelial growth factor (VEGF) mRNA and protein expression. The expression of endothelial cell markers CD31, VEGF receptor 2 (Flk-1) and eNOS as well as NO production were also markedly decreased. Functional studies showed HIF-1alpha knockdown via adenoviral siRNA transfer inhibited EPC colony formation, differentiation, proliferation and migration. These data indicate that specific gene knockdown via adenoviral transfer of siRNA is feasible in EPCs, and the effect is long-lasting. Our findings raise the possibility that such long-term modified human EPCs may be used to treat hypoxic tumor metastases that are known to be resistant to conventional therapeutic regimes.

Adenoviridae↗

PubMed2005

Knockdown of survivin expression by small interfering RNA induces apoptosis in human breast carcinoma cell line MCF-7.

BACKGROUND & OBJECTIVE: survivin, a member of inhibitor of apoptosis protein (IAP) gene family, expresses in various human cancer tissues, and may facilitate tumor cell evasion from apoptosis, and promote aberrant mitotic progression. This study was to investigate cell proliferation and apoptosis status of human breast carcinoma cell line MCF-7 after knockdown of survivin. METHODS: Small interfering RNA was transfected into MCF-7 cells to inhibit expression of survivin. mRNA and protein levels of survivin were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Proliferation inhibition rate of MCF-7 cells was analyzed by MTT assay. Cell apoptosis was assayed by flow cytometry (FCM). RESULTS: The expression of survivin in siRNA-transfected group decreased by 64% in comparison to untransfected group. After treatment of different concentrations of siRNA, proliferation inhibition rate and apoptosis rate of MCF-7 cells were increased. The highest proliferation inhibition rate was 60.9%, and the highest apoptosis rate was 29.0% after treatment of 200 nmol/L of siRNA. CONCLUSION: survivin siRNA might be a useful therapeutic agent for the treatment of breast carcinoma.

Apoptosis↗

PubMed2005

Small interfering RNA for experimental cancer therapy.

RNA interference describes the recently discovered process of sequence-specific, post-transcriptional gene silencing that is initiated by double-stranded RNA molecules known as small interfering RNAs (siRNAs). siRNAs have an acceptable half-life in vitro, a predictable biodistribution profile similar to that of single-stranded antisense oligonucleotides (ASOs), and have repeatedly been more robust than ASO techniques in terms of consistency of transcript knockdown and threshold concentration. Following validation in mammalian cells by Tuschl and co-workers in 2001, synthetic siRNAs have gained wide acceptance as a laboratory tool for target validation. Currently, there is considerable interest in the therapeutic use of siRNA, particularly in areas of infectious disease and cancer. In vitro and in vivo findings demonstrate the efficacy of siRNA knockdown of gene messages that are pivotal for tumor cell growth, metastasis, angiogenesis and chemoresistance, leading to tumor growth suppression. However, siRNA-based cancer therapy faces similar pharmacokinetic limitations to ASO therapy with respect to the extent that siRNA accesses primary and metastatic target cells. The recently identified 'off-target activity' of siRNAs is also of concern. The concept of carrier-restricted delivery of siRNA by conditionally replicative, oncolytic adenoviruses is discussed. Oncolytic adenoviral delivery offers the potential benefits of restricted and renewable siRNA expression within the tumor microenvironment, an additive antitumor outcome through viral oncolysis and siRNA-mediated oncogene silencing, and a proven clinical platform with respect to infectivity and safety.

Adenoviridae↗

PubMed2003

Evidence that TRPC1 (transient receptor potential canonical 1) forms a Ca(2+)-permeable channel linked to the regulation of cell volume in liver cells obtained using small interfering RNA targeted against TRPC1.

The TRPC1 (transient receptor potential canonical 1) protein, which is thought to encode a non-selective cation channel activated by store depletion and/or an intracellular messenger, is expressed in a number of non-excitable cells. However, the physiological functions of TRPC1 are not well understood. The aim of these studies was to investigate the function of TRPC1 in liver cells using small interfering RNA (siRNA) to ablate the TRPC1 protein. Treatment of H4-IIE liver cells with siRNA targeted against TRPC1 caused an approx. 50% decrease in expression of the human TRPC1 protein in cells transfected with cDNA encoding human TRPC1, and a 50% decrease in expression of the endogenous TRPC1 protein (assessed by Western blot and immunofluorescence). The decrease in endogenous TRPC1 protein in cells transfected with TRPC1 siRNA was associated with a greater increase in cell volume (compared with the increase observed in control cells) immediately after cells were placed in a hypotonic medium, and an enhanced regulatory cell volume decrease after exposure to hypotonic medium. Treatment with siRNA targeted against TRPC1 also led to a 25% inhibition of thapsigargin-stimulated Ca(2+) inflow, a 40% inhibition of ATP and maitotoxin-stimulated Ca(2+) inflow, and a 50% inhibition of maitotoxin-stimulated Mn(2+) inflow. The idea that, in liver cells, TRPC1 encodes a non-selective cation channel involved directly or indirectly in the regulation of cell volume is consistent with the results obtained.

Animals↗

PubMed2002

Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis.

RNA interference can be considered as an antisense mechanism of action that utilizes a double-stranded RNase to promote hydrolysis of the target RNA. We have performed a comparative study of optimized antisense oligonucleotides designed to work by an RNA interference mechanism to oligonucleotides designed to work by an RNase H-dependent mechanism in human cells. The potency, maximal effectiveness, duration of action, and sequence specificity of optimized RNase H-dependent oligonucleotides and small interfering RNA (siRNA) oligonucleotide duplexes were evaluated and found to be comparable. Effects of base mismatches on activity were determined to be position-dependent for both siRNA oligonucleotides and RNase H-dependent oligonucleotides. In addition, we determined that the activity of both siRNA oligonucleotides and RNase H-dependent oligonucleotides is affected by the secondary structure of the target mRNA. To determine whether positions on target RNA identified as being susceptible for RNase H-mediated degradation would be coincident with siRNA target sites, we evaluated the effectiveness of siRNAs designed to bind the same position on the target mRNA as RNase H-dependent oligonucleotides. Examination of 80 siRNA oligonucleotide duplexes designed to bind to RNA from four distinct human genes revealed that, in general, activity correlated with the activity to RNase H-dependent oligonucleotides designed to the same site, although some exceptions were noted. The one major difference between the two strategies is that RNase H-dependent oligonucleotides were determined to be active when directed against targets in the pre-mRNA, whereas siRNAs were not. These results demonstrate that siRNA oligonucleotide- and RNase H-dependent antisense strategies are both valid strategies for evaluating function of genes in cell-based assays.

Animals↗

PubMed2005

NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells.

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

Apoptosis↗

PubMed2003

Functional inhibition of the p75 receptor using a small interfering RNA.

The neurotrophin receptor p75(NTR) mediates a wide variety of biological effects. Consistent with the function in controlling the survival and neurite formation, p75(NTR) is expressed during the developmental stages of the nervous system. Importantly, p75(NTR) is re-expressed in various pathological conditions and is suggested to contribute to the inhibition of neuronal regeneration and the death of the neurons. Here we develop a tool to knock down the expression of p75(NTR) by employing a small interfering RNA (siRNA). The siRNA for p75(NTR) effectively reduces the expression of endogenous p75(NTR) both in Schwann cells and dorsal root ganglion neurons in vitro. NGF-induced cell death in Schwann cells and the neurite retraction in DRG neurons induced by myelin-associated glycoprotein are attenuated by the siRNA. Inhibition of p75(NTR) in specific pathological conditions by the siRNA may provide a potential therapeutic agent.

Animals↗

PubMed2006

p27 small interfering RNA induces cell death through elevating cell cycle activity in cultured cortical neurons: a proof-of-concept study.

Recent research has demonstrated that cell cycle-associated molecules are activated in multiple forms of cell death in mature neurons, and raised a hypothesis that unscheduled cell cycle activity leads to neuronal cell death. But there is little evidence that changes in endogenous level of these molecules are causally associated with neuronal cell death. Here we transfected small interfering RNA (siRNA) targeting cyclin-dependent kinase (CDK) inhibitor p27, which plays an important role in cell cycle arrest at G1-S phase, into cultured cortical neurons. Transfection of p27 siRNA reduced neuronal viability in a time-dependent manner. p27 siRNA induced phosphorylation of retinoblastoma protein (Rb), a marker of cell cycle progression at late G1 phase. Moreover, phosphorylation of Rb and neuronal cell death provoked by p27 siRNA were abrogated by pharmacological CDK inhibitors, olomoucine and purvalanol A. Our data demonstrate that a decrease in endogenous p27 induces neuronal cell death through elevating cell cycle activity.

Animals↗

PubMed2006

Effect of small interfering RNA targeting survivin gene on biological behaviour of bladder cancer.

BACKGROUND: Bladder cancer is the most common type of urinary system tumours. It is frequently associated with genetic mutations that deregulate the cell cycle and render these tumours resistant to apoptosis. Survivin, a newly discovered member inhibitor of apoptosis protein (IAP) family in several human cancers, by inducing cell proliferation and inhibiting apoptosis is frequently activated in bladder cancer. We studied the influence of small interfering RNA (siRNA) targeting survivin on the biological behaviour of bladder cancer cells. METHODS: A double strand survivin target sequence specific siRNA was designed and synthesized. After transfection of bladder cancer cell line T24 by siRNA/liposome complex with increasing concentrations (50200 nmol/L), the transfectant cells were intratumourally injected at different doses (5 microg or 50 microg). The effects were measured in vitro and in vivo. RESULTS: The selected siRNA efficiently down-regulated survivin mRNA expression in a dose and time dependent manner. The maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down-regulated by 75.91%. The inhibition rate of cell growth was 55.29% (P < 0.01) and the markedly increased apoptotic rate was 45.70% (P < 0.01). In vivo intratumoural injection of 50 microg siRNA-survivin could notably prevent the growth of bladder cancer (P < 0.01) in xenografted animals. CONCLUSION: The application of siRNA-survivin could markedly inhibit survivin expression in bladder cancer cell line by inducing apoptosis and inhibiting the growth of the tumour. It may become a new gene therapy tool for bladder cancer.

Animals↗

PubMed2007

An effective method for adenoviral-mediated delivery of small interfering RNA into mesenchymal stem cells.

Mesenchymal stem cells (MSCs) promise as a main actor of cell-based therapeutic strategies, due to their intrinsic ability to differentiate along different mesenchymal cell lineages, able to repair the diseased or injured tissue in which they are localized. The application of MSCs in therapies requires an in depth knowledge of their biology and of the molecular mechanisms leading to MSC multilineage differentiation. The knockdown of target genes through small interfering RNA (siRNA) carried by adenoviruses (Ad) represents a valid tool for the study of the role of specific molecules in cell biology. Unfortunately, MSCs are poorly transfected by conventional Ad serotype 5 (Ad5) vectors. We set up a method to obtain a very efficient transduction of rat MSCs with low doses of unmodified Ad5, carrying the siRNA targeted against the mRNA coding for Rb2/p130 (Ad-siRNA-Rb2), which plays a fundamental role in cell differentiation. This method allowed a 95% transduction rate of Ad-siRNA in MSC, along with a siRNA-mediated 85% decrease of Rb2/p130 mRNA and a 70% decrease of Rb2/p130 protein 48 h after transduction with 50 multiplicities of infection (MOIs) of Ad5. The effect on Rb2/p130 protein persisted 15 days after transduction. Finally, Ad-siRNA did not compromise the viability of transduced MSCs neither induced any cell cycle modification. The effective Ad-siRNA-Rb2 we constructed, together with the efficient method of delivery in MSCs we set up, will allow an in depth analysis of the role of Rb2/p130 in MSC biology and multilineage differentiation.

Adenoviridae↗

PubMed2005

Novel cationic cardiolipin analogue-based liposome for efficient DNA and small interfering RNA delivery in vitro and in vivo.

Cationic liposomes have been successfully used as an alternative approach to viral systems to deliver nucleic acids. However, high toxicity and inconsistent transfection efficiency have been associated with the currently available liposomes. Therefore, a novel cationic liposome was developed based on a synthetic cationic cardiolipin analogue (CCLA) to test the DNA transfection efficiency. This CCLA-based liposome was also used to determine the therapeutic efficacy of c-raf small interfering RNA (siRNA) in mice. In this report, we showed that the CCLA-based liposome was less toxic and effectively transfected reporter genes in vitro and in vivo. The transfection efficiency in mice was seven-fold higher than the commercially available DOTAP-based liposome. In addition, c-raf siRNA in the presence of CCLA-based liposome induced up to 62% of growth inhibition in cancer cells. Treatment of c-raf siRNA/CCLA complex in SCID mice bearing human breast xenograft tumors resulted in 73% of tumor growth suppression as compared to free c-raf siRNA group. In conclusion, a novel CCLA-based liposome showed less toxicity and broad usage both in vitro and in vivo with DNA and siRNA.

Animals↗

PubMed2007

Specific beta1-adrenergic receptor silencing with small interfering RNA lowers high blood pressure and improves cardiac function in myocardial ischemia.

OBJECTIVES: Beta-blockers are widely used and effective for treating hypertension, acute myocardial infarction (MI) and heart failure, but they present side-effects mainly due to antagonism of beta2-adrenergic receptor (AR). Currently available beta-blockers are at best selective but not specific for beta1 or beta2-AR. METHODS: To specifically inhibit the expression of the beta1-AR, we developed a small interfering RNA (siRNA) targeted to beta1-AR. Three different sequences of beta1 siRNA were delivered into C6-2B cells with 90% efficiency. RESULTS: One of the three sequences reduced the level of beta1-AR mRNA by 70%. The siRNA was highly specific for beta1-AR inhibition with no overlap with beta2-AR. To test this in vivo, systemic injection of beta1 siRNA complexed with liposomes resulted in efficient delivery into the heart, lung, kidney and liver, and effectively reduced beta1-AR expression in the heart without altering beta2-AR. beta1 siRNA significantly lowered blood pressure of spontaneously hypertensive rats (SHR) for at least 12 days and reduced cardiac hypertrophy following a single injection. Pretreatment with beta1 siRNA 3 days before induction of MI in Wistar rats significantly improved cardiac function, as demonstrated by dP/dt and electrocardiogram following the MI. The protective mechanism involved reduction of cardiomyocyte apoptosis in the beta1 siRNA-treated hearts. CONCLUSIONS: The present study demonstrates the possibility of using siRNA for treating cardiovascular diseases and may represent a novel beta-blocker specific for beta1-AR.

Animals↗

PubMed2006

Attenuation of collagen production with small interfering RNA of SPARC in cultured fibroblasts from the skin of patients with scleroderma.

OBJECTIVE: Recently, it was observed that SPARC (secreted protein, acidic and rich in cysteine) is overexpressed in the fibroblasts of skin biopsy specimens obtained from patients with systemic sclerosis (SSc; scleroderma), and that specific inhibition of SPARC expression in normal human fibroblasts attenuated the profibrotic effect of transforming growth factor beta (TGFbeta). The purpose of this study was to examine whether inhibition of SPARC with small interfering RNA (siRNA) can be used to ameliorate the overproduction of major extracellular matrix components in SSc fibroblasts. METHODS: Fibroblasts obtained from biopsy specimens of the unaffected skin of 3 patients with diffuse SSc and 3 age- and sex-matched healthy controls were cultured and transfected with SPARC siRNA. Before and after transfection, real-time quantitative reverse transcription-polymerase chain reaction, immunostaining, and Western blotting were used to examine the transcription and/or protein levels of SPARC, connective tissue growth factor (CTGF), type I collagen, type II collagen, TGFbeta receptor 1 (TGFbetaR1), and Smad3. Student's paired t-tests were used to determine the significance of the results. RESULTS: SSc fibroblasts showed overexpression of SPARC, COL1A2, COL3A1, and CTGF, which is consistent with an activated fibroblast phenotype. Upon inhibition of SPARC with siRNA, these activated SSc fibroblasts showed decreased gene expression of COL1A2, COL3A1, and CTGF (43%, 54%, and 58%, respectively). In addition, the overproduction of type I collagen protein in SSc fibroblasts was inhibited and was shown to parallel levels of SPARC expression. In contrast, the expression of TGFBR1 and Smad3 did not change significantly nor was the level of phosphorylation of Smad3 reduced upon SPARC siRNA silencing in unstimulated cultured SSc fibroblasts. CONCLUSION: Overproduction of collagens in SSc skin fibroblasts can be attenuated through SPARC silencing. This inhibition may be associated either with direct interaction between SPARC and collagens or with inhibition of CTGF, which is a downstream effector of TGFbeta signaling. However, SPARC silencing in SSc fibroblasts appears not to be associated with TGFBR1- and Smad3-dependent processes. Application of SPARC silencing represents a potential therapeutic approach to SSc.

Activin Receptors, Type I↗

PubMed2006

Pharmacological inhibition or small interfering RNA targeting acid ceramidase sensitizes hepatoma cells to chemotherapy and reduces tumor growth in vivo.

Ceramidases (CDases) play a key role in cancer therapy through enhanced conversion of ceramide into sphingosine 1-phosphate (S1P), but their involvement in hepatocarcinogenesis is unknown. Here, we report that daunorubicin (DNR) activated acid CDase post-transcriptionally in established human (HepG2 cells) or mouse (Hepa1c1c7) hepatoma cell lines as well as in primary cells from murine liver tumors, but not in cultured mouse hepatocytes. Acid CDase silencing by small interfering RNA (siRNA) or pharmacological inhibition with N-oleoylethanolamine (NOE) enhanced the ceramide to S1P balance compared to DNR alone, sensitizing hepatoma cells (HepG2, Hep-3B, SK-Hep and Hepa1c1c7) to DNR-induced cell death. DNR plus NOE or acid CDase siRNA-induced cell death was preceded by ultrastructural changes in mitochondria, stimulation of reactive oxygen species generation, release of Smac/DIABLO and cytochrome c and caspase-3 activation. In addition, in vivo siRNA treatment targeting acid CDase reduced tumor growth in liver tumor xenografts of HepG2 cells and enhanced DNR therapy. Thus, acid CDase promotes hepatocarcinogenesis and its antagonism may be a promising strategy in the treatment of liver cancer.

Animals↗

PubMed2006

Small interfering RNA targeting epidermal growth factor receptor enhances chemosensitivity to cisplatin, 5-fluorouracil and docetaxel in head and neck squamous cell carcinoma.

Overexpression of epidermal growth factor receptor (EGFR) has been found in various epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC), and is associated with increased tumor growth, metastasis, resistance to chemotherapeutic agents and poor prognosis. As such, EGFR is a potential target for antitumor therapy and several EGFR inhibitors have been investigated in preclinical or clinical settings. In the present study, we used small interfering RNA (siRNA) to downregulate EGFR expression while evaluating the effect of EGFR siRNA on cell proliferation, and the combined effects with cisplatin, 5-fluorouracil (5-FU) and docetaxel in HNSCC. Furthermore, HNSCC xenografts were treated with EGFR siRNA alone or in combination with cisplatin, and tumor growth was examined. EGFR expression, proliferation, angiogenesis and apoptosis index were evaluated by immunohistochemistry. The results showed that EGFR siRNA efficiently downregulated EGFR expression and inhibited cell growth of HNSCC. Treatment with EGFR siRNA in combination with cisplatin, 5-FU and docetaxel enhanced chemosensitivity with a significant increase in apoptosis. EGFR siRNA delivered by atelocollagen enhanced the antitumor effect of cisplatin in the HNSCC xenograft model. These cumulative results suggest that EGFR siRNA combined with cisplatin, 5-FU and docetaxel may be a feasible strategy to enhance the effects of chemotherapy in patients with HNSCC.

Animals↗

PubMed2005

Knockdown of the Dnmt1s transcript using small interfering RNA in primary murine and bovine fibroblast cells.

RNA interference (RNAi) has rapidly developed into one of the most widely applied technologies in molecular and cellular research, and although young, is now an essential experimental tool. The versatility of RNAi, especially in mammalian species, lends to its potential applications in a wide array of fields. Without having to genetically manipulate the genome, the ability to selectively reduce the level of a specific transcript using small interfering RNA (siRNA) molecules has great appeal in studying reprogramming issues in somatic cell nuclear transfer (SCNT) embryos. In such embryos, the aberrant expression of the somatic isoform of Dnmt1 (Dnmt1s), the enzyme responsible for maintaining DNA methylation in all somatic cells, has been implicated as one factor in the improper reprogramming of the donor genome. In the present study, the ability to develop a method allowing for the knockdown, or reduction, of Dnmt1s in primary fibroblast cells, like those commonly used as karyoplast donors in SCNT studies, was investigated in primary murine and bovine fibroblast cells as well as in a compromised cell line (NIH/3T3). Two Dnmt1s-specific siRNA candidates were designed and tested. Using optimized conditions, these siRNAs were transiently transfected into the cells with total RNA and nuclear protein being collected. A 56.5% knockdown in Dnmt1s was achieved in the compromised and primary murine cells whereas Dnmt1s was reduced by 15.4% in the primary bovine cells. A reduction in Dnmt1s mRNA did not correspond to a reduction in protein as determined by immunodetection of Western blots. Overall, this study demonstrated the ability of siRNA to knockdown Dnmt1s mRNA in primary fibroblast donor cells. In order to substantially increase the efficiency while decreasing the anomalies seen in SCNT, novel techniques, like the one proposed, are needed to assist the oocyte's ability to reprogram a differentiated genome.

Animals↗