Killing the messenger: short RNAs that silence gene expression

Dykxhoorn, Derek M.; Novina, Carl D.; Sharp, Phillip A.

doi:10.1038/nrm1129

Cited by 1,063 publications

(730 citation statements)

References 112 publications

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“…30 The suppression of oncogenic N-Ras in melanoma cells harboring this mutation did not appear to be entirely specific, as siRNA directed at WT NRAS in cells harboring only mutant NRAS also resulted in a small reduction (25%) in N-Ras expression. Furthermore, we observed that targeting WT NRAS using siRNA directed at oncogenic NRAS resulted in decreased N-Ras expression in melanoma cells with only WT NRAS alleles.…”

Section: Discussionmentioning

confidence: 97%

Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells

Eskandarpour¹,

Kiaii²,

Zhu³

et al. 2005

Intl Journal of Cancer

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The majority of human melanomas harbor activating mutations in either the BRAF or NRAS gene. To date, the role of oncogenic NRAS in melanoma remains poorly defined and no current therapies are directed at specifically suppressing oncogenic NRAS in human melanoma tumors. The aim of our study, therefore, was to investigate the effects of suppressing oncogenic NRAS in human melanoma cell lines in vitro. Using both small interfering RNAand plasmid based-RNA interference techniques, oncogenic NRAS was specifically suppressed in 2 human melanoma cell lines, 224 and BL, which harbor a codon 61 CAA (glutamine) to CGA (arginine) NRAS mutation. Suppression of oncogenic NRAS in these cell lines resulted in increased apoptosis. Furthermore, in 224 cells we demonstrated decreased phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, and reduced expression of NF-kB and cyclin D1 in the N-Ras signaling pathway. In contrast, RNA interference directed at wild-type (WT) NRAS had no significant effect on apoptosis of 224 cells or 2 human melanoma cell lines (A375 and 397) containing WT NRAS but a codon 600 GTG (valine) to GAG (glutamate) mutation in BRAF. These data suggest that oncogenic NRAS is important for avoidance of apoptosis in melanomas that harbor the codon 61 NRAS mutation and emphasizes oncogenic NRAS as a therapeutic target in patients with tumors that harbor this mutation. ' 2005 Wiley-Liss, Inc.Key words: RNAi; melanoma; Ras; signal transduction; apoptosis Activation of Ras is a common molecular event in the etiology of human cancer. 1 The Ras proteins H-, K-and N-Ras are central regulators of cellular signal transduction processes leading to cell growth and differentiation. In the resting cell, Ras proteins are in the guanosine diphosphate (GDP)-bound inactive state. However, in response to receptor protein tyrosine kinases or other growth factor-dependent stimuli, these Ras proteins become activated by binding guanosine triphosphate (GTP). 2,3 Oncogenic mutations in codons 12, 13 and 61 of RAS genes prevent GTP hydrolysis, resulting in constitutively active Ras proteins. Such mutations that change the protooncogene RAS to an active oncogene are found in approximately 30 to 50% of human tumors. [4][5][6][7][8] Ras has also been demonstrated to be important for both the genesis and maintenance of melanoma. For instance, in a doxycycline-inducible H-Ras V12G mouse melanoma model, withdrawal of doxycycline was demonstrated to result in histological regression of primary and implanted tumors accompanied by marked apoptosis of melanoma cells. 9 Studies on melanoma tumors have confirmed that oncogenic activation of NRAS constitutes the predominant RAS alteration in cutaneous melanoma, with NRAS codon 61 mutations being the most common alterations and codon 12 and 13 mutations being less frequent. 6,7,10,11 In sporadic melanoma cases, the frequency of NRAS alterations reported varies in different studies. [12][13][14] In a study performed by our group, 28% of primary melanomas and 37% of metastatic tumo...

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Section: Discussionmentioning

confidence: 97%

Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells

Eskandarpour¹,

Kiaii²,

Zhu³

et al. 2005

Intl Journal of Cancer

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“…There is currently substantial hope that RNA interference (RNAi)-mediated modulation of protein expression can be used for prevention or treatment of diseases, 1 but only a few studies in whole animals have been reported so far. Here, we show that transgenic expression of a short hairpin (sh) RNA targeting Bim efficiently prevents the development of polycystic kidney disease in Bcl-2-deficient mice and that this effect can be transmitted over several generations.…”

Section: Dear Editormentioning

confidence: 99%

Polycystic kidney disease prevented by transgenic RNA interference

Bouillet¹,

Robati²,

Bath³

et al. 2005

Cell Death Differ

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“…The mechanism of RNAi is that the short double-stranded (ds) RNA can mediate the degradation of sequence specific mRNA [38][39][40] . It has been reported that introducing small interfering RNAs (siRNAs) or short hairpin RNA (shRNA) into mammalian cells could specifically silence the expression of target genes [41][42][43] .…”

Section: Introductionmentioning

confidence: 99%

RNA interference mediated silencing of α-synuclein in MN9D cells and its effects on cell viability

et al. 2008

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ObjectiveTo silence the expression of -synuclein in MN9D dopaminergic cells using vector mediated RNA interference (RNAi) and examined its effects on cell proliferation and viability. Methods We identified two 19-nucleotide stretches within the coding region of the -synuclein gene and designed three sets of oligonucleotides to generate doublestranded (ds) oligos. The ds oligos were inserted into the pENTR TM /H1/TO vector and transfected into MN9D dopaminergic cells. -Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-diphenytetrazoliumromide (MTT). Results The mRNA and protein levels of -synuclein gene were significantly down-regulated in pSH2/ -SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSH1/ -SYNtransfected cells showed no significant difference. Silencing -synuclein expression does not affect cell proliferation but may decrease cell viability. Conclusion Our results demonstrated pSH2/ -SYN is an effective small interfering RNA (siRNA) sequence and potent silencing of mouse -synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of -synuclein and examining its role in Parkinson's disease (PD) pathogenesis.-Synuclein may be important for the viability of MN9D cells, and loss of -synuclein may induce cell injury directly or indirectly.

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Killing the messenger: short RNAs that silence gene expression

Cited by 1,063 publications

References 112 publications

Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells

Suppression of oncogenic NRAS by RNA interference induces apoptosis of human melanoma cells

Polycystic kidney disease prevented by transgenic RNA interference

RNA interference mediated silencing of α-synuclein in MN9D cells and its effects on cell viability

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