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Palo Alto, CA, United States

Noonan E.J.,Center for Molecular Biology in Medicine
Oncotarget | Year: 2010

MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNAs) that regulate gene expression by repressing translation or triggering the degradation of complementary mRNA sequences. Certain miRNAs have been shown to function as integral components of the p53 and/or retinoblastoma (Rb) regulatory networks. As such, miRNA dysregulation can have a profound effect on cancer development. Previous studies have shown that miR-449a is down-regulated in human prostate cancer tissue and possesses potential tumor suppressor function. In the present study, we identify miR-449a-mediated growth arrest in prostate cancer cells is dependent on the Rb protein. We show that mutant Rb prostate cancer cells (DU- 145) are resistant to cell cycle arrest and cellular senescence induced by miR- 449a, while overexpression of wild-type Rb in DU-145 sublines (DU-1.1 and B5) restores miR-449a function. In silico analysis of 3'UTR regions reveal a putative miR-449a target site in the transcript of Cyclin D1 (CCND1); an oncogene involved in directly regulating Rb activity and cell cycle progression. Luciferase 3'UTR reporter constructs and inhibitory oligonucleotides confirm that Cyclin D1 is a direct downstream target of miR-449a. We also reveal that miR-449a suppresses Rb phosphorylation through the knockdown of Cyclin D1 and previously validated target HDAC1. By targeting genes involved in controlling Rb activity, miR- 449a regulates growth and senescence in an Rb-dependent manner. These data indicate that miR-449a is a miRNA component of the Rb pathway and its tumor suppressor-like effects, in part, depends on Rb status in prostate cancer cells.

Place R.F.,University of California at San Francisco | Noonan E.J.,Center for Molecular Biology in Medicine | Noonan E.J.,Stanford University | Foldes-Papp Z.,Medical University of Graz | Li L.-C.,University of California at San Francisco
Current Pharmaceutical Biotechnology | Year: 2010

RNA interference (RNAi) is an evolutionary conserved mechanism by which small double-stranded RNA (dsRNA) - termed small interfering RNA (siRNA) - inhibit translation or degrade complementary mRNA sequences. Identifying features and enzymatic components of the RNAi pathway have led to the design of highly-effective siRNA molecules for laboratory and therapeutic application. RNA activation (RNAa) is a newly discovered mechanism of gene induction also triggered by dsRNAs termed small activating RNA (saRNA). It offers similar benefits as RNA interference (RNAi), while representing a new method of gene overexpression. In the present study, we identify features of RNAa and explore chemical modifications to saRNAs that improve the applicability of RNAa. We evaluate the rate of RNAa activity in order to define an optimal window of gene induction, while comparing the kinetic differences between RNAa and RNAi. We identify Ago2 as a conserved enzymatic component of both RNAa and RNAi implicating that saRNA may tolerate modification based on Ago2 function. As such, we define chemical modifications to saRNAs that manipulate RNAa activity, as well as exploit their effects to design saRNAs with enhanced medicinal properties. These findings reveal functional features of RNAa that may be utilized to augment saRNA function for mechanistic studies or the development of RNAa-based drugs. © 2010 Bentham Science Publishers Ltd.

Place R.F.,University of California at San Francisco | Place R.F.,RNA Therapeutics Inc. | Wang J.,University of California at San Francisco | Noonan E.J.,Center for Molecular Biology in Medicine | And 8 more authors.
Molecular Therapy - Nucleic Acids | Year: 2012

Application of RNA interference (RNAi) in the clinic has improved with the development of novel delivery reagents (e.g., lipidoids). Although RNAi promises a therapeutic approach at silencing gene expression, practical methods for enhancing gene production still remain a challenge. Previously, we reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a phenomenon termed RNA activation (RNAa). In the present study, we investigate the therapeutic potential of RNAa in prostate cancer xenografts by using lipidoid-based formulation to facilitate in vivo delivery. We identify a strong activator of gene expression by screening several dsRNAs targeting the promoter of tumor suppressor p21WAF1/ Cip1 (p21). Chemical modification is subsequently implemented to improve the medicinal properties of the candidate duplex. Lipidoid-encapsulated nanoparticle (LNP) formulation is validated as a delivery vehicle to mediate p21 induction and inhibit growth of prostate tumor xenografts grown in nude mice following intratumoral injection. We provide insight into the stepwise creation and analysis of a putative RNAa-based therapeutic with antitumor activity. Our results provide proof-ofprinciple that RNAa in conjunction with lipidioids may represent a novel approach for stimulating gene expression in vivo to treat disease © 2012 American Society of Gene and Cell Therapy All rights reserved.

Xiang H.,Center for Molecular Biology in Medicine | Xiang H.,Stanford University | Noonan E.J.,Center for Molecular Biology in Medicine | Noonan E.J.,Stanford University | And 9 more authors.
Leukemia | Year: 2011

Human follicular B-cell lymphoma is associated with the t(14;18) chromosomal translocation that juxtaposes the Bcl2 proto-oncogene with the immunoglobulin heavy chain (Igh) locus, resulting in the deregulated expression of Bcl2. Our previous studies have shown that the Igh 3′ enhancers deregulate the Bcl2 expression in vitro. However, the effects of the Igh 3′ enhancer elements on Bcl2 expression in vivo are not known. To investigate the role of the Igh 3′ enhancers in Bcl2 deregulation, we used gene targeting to generate knock-in mice in which four DNase I-hypersensitive regions from the murine Igh 3′ region were integrated 3′ of the Bcl2 locus. Increased levels of Bcl2 mRNA and protein were observed in the B cells of Igh-3′E-bcl2 mice. B cells from Igh-3′E-bcl2 mice showed an extended survival in vitro compared with B cells from wild-type (Wt) mice. The Bcl2 promoter shift from P1 (the 5′ promoter) to P2 (the 3′ promoter) was observed in B cells from Igh-3′E-bcl2 mice, similar to human t(14;18) lymphomas. The IgH-3′E-bcl2 mice developed monoclonal B-cell follicular lymphomas, which were slowly progressive. These studies show that the Igh 3′ enhancers have an important role in the deregulation of Bcl2 and B-cell lymphomagenesis in vivo. © 2011 Macmillan Publishers Limited All rights reserved.

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