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Results for “RNAi”

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At least 721 records · Page 40Linked to original sources
PubMed2005

Polo-like kinase-1: activity measurement and RNAi-mediated knockdown.

Polo-like kinase-1 (Plk-1) is an important cell cycle regulatory kinase that has been implicated in a multitude of cell cycle events. In this chapter we review those multiple functions of Plk-1 and describe the methods routinely used in our laboratory to purify Plk-1 from cellular lysates and measure Plk-1 kinase activity in vitro. In addition, we describe a method to analyze cell cycle progression after depletion of Plk-1 by RNA-interference in tissue culture cells.

Animals↗
PubMed2006

Antiviral applications of RNAi for coronavirus.

Until the appearance of severe acute respiratory syndrome (SARS), caused by the SARS coronavirus (SARS-CoV) in early 2003, coronavirus infection was not considered to be serious enough to be controlled by either vaccination or specific antiviral therapy. It is now believed that the availability of antiviral drugs effective against SARS-CoV will be crucial for the control of future SARS outbreaks. Recently, RNA interference has been successfully used as a more specific and efficient method for gene silencing. RNA interference induced by small interfering RNA can inhibit the expression of viral antigens and so provides a new approach to the therapy of pathogenic viruses. This review provides an overview of current information on coronavirus and the application of small interfering RNA in viral therapeutics, with particular reference to SARS-CoV.

Animals↗
PubMed2004

Therapeutic potential of retroviral RNAi vectors.

The ability of small interfering RNA (siRNA) to mediate gene-specific post-transcriptional silencing in mammalian cells will undoubtedly revolutionise functional genomics, as well as drug target identification and validation. Furthermore, there is widespread excitement that siRNA itself might prove useful in the clinical setting. For those wishing to develop siRNA as a therapeutic agent, the most difficult obstacle to overcome will be delivery. Recently, several breakthroughs have highlighted viruses as excellent vehicles for siRNA delivery. Retroviruses, the transgene-delivery vector of choice for many experimental gene therapy studies, have been engineered to deliver and stably express therapeutic siRNA within cells, both in vitro and in vivo. These findings are important milestones for the development of siRNA as a gene therapy for treatment of viral infections, cancer, autoimmune syndromes and numerous genetic disorders. This review describes the development of retroviral siRNA vectors, highlights proof-of-concept experiments demonstrating their therapeutic efficacy and explores therapeutic targets particularly suitable for retroviral-mediated gene silencing.

Animals↗
PubMed2004

RNAi and siRNA.

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Drug Delivery Systems↗
PubMed2004

Hepatic PTP-1B expression regulates the assembly and secretion of apolipoprotein B-containing lipoproteins: evidence from protein tyrosine phosphatase-1B overexpression, knockout, and RNAi studies.

Protein tyrosine phosphatase-1B (PTP-1B) plays an important role in regulation of insulin signal transduction, and modulation of PTP-1B expression seems to have a profound effect on insulin sensitivity and diet-induced weight gain. The molecular link between PTP-1B expression and metabolic dyslipidemia, a major complication of insulin resistance, was investigated in the present study using PTP-1B knockout mice as well as overexpression and suppression of PTP-1B. Chronic fructose feeding resulted in a significant increase in plasma VLDL in wild-type mice but not in PTP-1B knockout mice. Lipoprotein profile analysis of plasma from PTP-1B knockout mice revealed a significant reduction in apolipoprotein B (apoB100) lipoproteins, associated with reduced hepatic apoB100 secretion from isolated primary hepatocytes. In addition, treatment of cultured hepatoma cells with PTP-1B siRNA reduced PTP-1B mass by an average of 41% and was associated with a 53% decrease in secretion of metabolically labeled apoB100. Conversely, adenoviral-mediated overexpression of PTP-1B in HepG2 cells downregulated the phosphorylation of insulin receptor and insulin receptor substrate-1 and caused increases in cellular and secreted apoB100 as a result of increased intracellular apoB100 stability. Collectively, these findings suggest that PTP-1B expression level is a key determinant of hepatic lipoprotein secretion, and its overexpression in the liver can be sufficient to induce VLDL overproduction and the transition to a metabolic dyslipidemic state.

Animals↗
PubMed2005

Analysis of metastasis suppressing function of E-cadherin in gastric cancer cells by RNAi.

AIM: To study the effect of inhibited E-cadherin expression on invasion of cancer cells. METHODS: We designed the nucleotide sequence of siRNA corresponding to 5' non-coding and coding sequence of E-cadherin. 21-nucleotide dssiRNA was synthesized by in vitro transcription with Ambion Silencer TM siRNA Construction Kit. siRNA was transfected into gastric cancer MKN45 using TransMessenger transfection Kit. RT-PCR and immunofluorescent assay were used to investigate the inhibition of the expression of mutated E-cadherin. Invasive ability of cancer cells was determined by Transwell assay. RESULTS: The synthesis of E-cadherin mRNA rather than protein expression was suppressed dramatically 7 d after interference. Decreased protein expression was observed on d 10 after interference. On d 11, invasion ability was enhanced significantly. CONCLUSION: siRNA targeted at non-coding and coding sequence of E-cadherin showed significant inhibition on mRNA and protein expression. Inhibited E-cadherin expression results in increased invasion ability of cancer cells.

Adenocarcinoma↗
PubMed2005

Downregulation of survivin by RNAi inhibits the growth of esophageal carcinoma cells.

Esophageal squamous cell carcinoma ranks among one of the most frequent cause of cancer death in the world. Understanding of the molecular mechanisms involved in the pathogenesis of esophageal cancer becomes critical to develop more effective treatments. Elevated expression of survivin in esophageal carcinoma has been reported before and suppression of survivin expression leads to many tumor cells growth inhibition. We hypothesized that downregulation of survivin would inhibit the growth of human esophageal cancer cells. RNA interference directed against survivin was introduced into a human esophageal squamous cell carcinoma cell line KYSE510. Stable clones were selected and western blot analysis was performed to detect the protein level of survivin. Tumor cell growth in vitro and in vivo was assessed by trypan blue exclusion and nude mice experiments. Annexin V/propidium iodide staining followed by flow cytometric analysis and TUNEL assay were used to detect apoptosis in cell culture and in nude mice. We found that RNA interference could efficiently and stably suppress survivin expression in KYSE510 cells. Downregulation of survivin resulted in significantly inhibition of tumor growth in vitro and in vivo. The mechanism appears to be increased induction of apoptosis. Our results suggest a potential role for the targeting of survivin in the treatments of esophageal carcinoma.

Apoptosis↗