RNA-mediated Interference Research Articles

Preparation of <em>Drosophila</em> S2 cells for Light Microscopy

The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells. S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24°C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks. Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is this: first, S2 cells (which can possess transgenes to express tagged markers) are treated by RNAi to eliminate a target protein(s). RNAi treatment time can be adjusted to allow for differences in protein turn-over kinetics and to minimize cell trauma/death if the target protein is important for viability. Next, the treated cells are transferred to a dish containing a coverslip pre-coated with conA to induce cells to spread and tightly adhere to the glass. Finally, cells are imaged with the researcher's choice of microscopy modes. S2 cells are particularly good for studies requiring extended visualization of live cells since these cells stay healthy at room temperature and normal atmosphere.

Synaptotagmin-mediated vesicle fusion regulates cell migration

Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release.

Effect of retroviral vector mediating RNA interference on the release of tumor necrosis factor-alpha in lipopolysaccharide-stimulating macrophage

To explore the effect of small interfering RNA (siRNA) targeting NF-kappaB signal pathway on the expression level of tumor necrosis factor alpha (TNF-alpha) released by lipopolysaccharides (LPS)-stimulating-macrophages. Human monocytic THP-1 cell was induced by phorbol myristate acetate (PMA) and transformed into macrophage. Two groups of macrophage were infected by siRNA retroviral expression vector specific to NF-kappaB functional subunit P65 (siRNA group) and Scramble control vector (Scramble control group) constructed by molecular cloning technology. Lipopolysaccharide (50 microg/ml) was used to treat the macrophages continuously. RT-PCR was performed to detect the expression level of NF-kappaB P65 mRNA and TNF-alpha mRNA at different time-points of LPS stimulation. Western blotting was used to analyze the protein level of NF-kappaB P65. Enzyme-linked immunosorbent assay was applied to analyze the expression level of TNF-alpha released by LPS-stimulated macrophages. At Hours 12 and 24 after LPS stimulation, the expression level of NF-kappaB P65 mRNA in siRNA group (0.97 +/- 0.02, 0.89 +/- 0.01) was significantly less than that in Scramble control group (1.01 +/- 0.03, 0.97 +/- 0.01, both P < 0.05). At Hours 24 and 72 after LPS stimulation, the expression level of NF-kappaB P65 protein in siRNA group (0.95 +/- 0.04, 0.94 +/- 0.01) was obviously less than that in Scramble control group (1.07 +/- 0.06, 1.03 +/- 0.05, both P < 0.05). At Hours 4, 8, 12 and 24 after LPS stimulation, TNF-alpha mRNA released by siRNA group macrophages was far less than that by Scramble control group macrophages (0.92 +/- 0.02 vs 0.98 +/- 0.01, 0.86 +/- 0.02 vs 1.00 +/- 0.01, 0.79 +/- 0.03 vs 1.01 +/- 0.01, 0.78 +/- 0.03 vs 1.02 +/- 0.01, all P < 0.05). At Hours 2, 4, 8, 24, 36, 48, 54 and 72 after LPS stimulation, the TNF-alpha content in culture medium supernatant in siRNA group macrophage was less than that in scramble control group (P < 0.05). NF-kappaB P65 siRNA inhibits the functional activity of NF-kappaB signal pathway in PMA-induced macrophage. Then it blocks the activation of macrophage and the excessive release of TNF-alpha due to endotoxin stimulation. The RNA interference technology may be applied to prevent and treat excessive inflammatory reaction in acute lung injury.

The nuclear export receptor XPO-1 supports primary miRNA processing in C. elegans and Drosophila

MicroRNA (miRNA) biogenesis proceeds from a primary transcript (pri-miRNA) through the pre-miRNA into the mature miRNA. Here, we identify a role of the Caenorhabditis elegans nuclear export receptor XPO-1 and the cap-binding proteins CBP-20/NCBP-2 and CBP-80/NCBP-1 in this process. The RNA-mediated interference of any of these genes causes retarded heterochronic phenotypes similar to those observed for animals with mutations in the let-7 miRNA or core miRNA machinery genes. Moreover, pre- and mature miRNAs become depleted, whereas primary miRNA transcripts accumulate. An involvement of XPO-1 in miRNA biogenesis is conserved in Drosophila, in which knockdown of Embargoed/XPO-1 or its chemical inhibition through leptomycin B causes pri-miRNA accumulation. Our findings demonstrate that XPO-1/Emb promotes the pri-miRNA-to-pre-miRNA processing and we propose that this function involves intranuclear transport and/or nuclear export of primary miRNAs.

Inhibitory effects of lentivirus mediated RNA interference targeting human Wip1 gene on the prolif-eration of human glioma U251 cells

Objective To construct the lentiviral expression vector for RNA interference (RNAi) of Wipl gene in glioma cells and provide research foundation for studying the mechanisms that wild-type p53-induced phosphatase 1 (Wip1 ) gene functions for malignant proliferation of gliomas. Methods Three effective sequences of RNAi targeting Wip1 were confirmed. Both sense and antisense Oligo DNA of the tar-geting sequence were designed, synthesized and cloned into the pFU-GW-iRNA vector. After cotransfectsd with lentiviral vector of T293 cells, the lentivirus was produced and the titer of virus was tested. U251 cells were infected with the lentivirus and the expression of Wip1 mRNA and protein in U251 cells was detected by real-time quantitative polymerase chain reaction (PCR) and Western blotting. The propagation of U251 was detected by CCK-8 and expression of bcl-2 was detected by Western blotting. Results A recombinant lentiviral vector expressing shRNAs against Wip1 gene was obtained and confirmed by DNA sequencing. The titer of virus was 3×10~8-8×10~8 TU/ml. Wipl mRNA and protein expression in U251 cells after infec-ted with lentiviral vector was decreased remarkably. The mRNA expression was 36. 3% ,32. 9% and 23.8% respectively, compared with the control group. And the lentiviral shRNA expression vector reduced cell pro-liferation by 35. 1% 4 d after stable Wipl gene knock-down in U251 cells. Conclusion The lentiviral shRNA expression vector targeting human Wipl gene capable of stable Wipl gene knock-down in U251 cells has been successfully constructed,which provides a basis for further study of mechanisms that Wip1 gene acta for malignant proliferation of gliomas. And preliminary conclution was drawn that Wipl gene may promote the growth of U251 cells. Key words: Wip1 gene; Glioma; RNA interference; Lentiviral

2134 STAPHYLOCOCCAL NUCLEASE DOMAIN-CONTAINING PROTEIN 1 AS A POTENTIAL TISSUE MARKER FOR PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Markers II1 Apr 20102134 STAPHYLOCOCCAL NUCLEASE DOMAIN-CONTAINING PROTEIN 1 AS A POTENTIAL TISSUE MARKER FOR PROSTATE CANCER Hidetoshi Kuruma, Yuko Kamata, Hiroyuki Takahashi, Koji Igarashi, Takahiro Kimura, Kenta Miki, Jun Miki, Hiroshi Sasaki, Norihiro Hayashi, and Shin Egawa Hidetoshi KurumaHidetoshi Kuruma Tokyo, Japan More articles by this author , Yuko KamataYuko Kamata Tokyo, Japan More articles by this author , Hiroyuki TakahashiHiroyuki Takahashi Tokyo, Japan More articles by this author , Koji IgarashiKoji Igarashi Kanagawa, Japan More articles by this author , Takahiro KimuraTakahiro Kimura Tokyo, Japan More articles by this author , Kenta MikiKenta Miki Tokyo, Japan More articles by this author , Jun MikiJun Miki Tokyo, Japan More articles by this author , Hiroshi SasakiHiroshi Sasaki Tokyo, Japan More articles by this author , Norihiro HayashiNorihiro Hayashi Tokyo, Japan More articles by this author , and Shin EgawaShin Egawa Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.2222AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Using high molecular-weight proteomic analysis, we previously showed that Staphylococcal nuclease domain-containing protein 1 (SND1) is highly expressed in recurrent androgen-insensitive prostate cancer tissues. SND1 is a component of the RNA-induced splicing complex that mediates RNA interference, leading to degradation of specific mRNAs. The objective of this study was to further characterize SND1 expression and to investigate its biological potential in prostate cancer. METHODS Radical prostatectomy specimens were obtained from 62 prostate cancer patients. SND1 immunohistochemical staining patterns were evaluated using an in-house polyclonal antibody. To assess the potential for tissue marker, immunostaining intensity of SND1 was compared with α-methylacyl-coenzyme A racemase (AMACR) that is a clinically applicant tissue marker protein. We confirmed SND1 mRNA expression in prostate cancer cells using an in situ hybridization technique. To determine the affection of SND1 for cell proliferation, we knocked down SND1 gene in prostate cancer PC-3 cells in vitro with small interfering RNA and observed cell growth with MTT assay. RESULTS In prostate cancer specimen, SND1 expressions were detected in 97%, whereas weakly or not at all expressed in all benign prostatic glands, including the hyperplastic glands and normal luminal cells. The immunostaining intensity scores in cancer, high-grade prostatic intraepithelial neoplasia (HGPIN), hyperplasia, and normal luminal cells were 1.7, 0.93, 0.24, and 0.27, (p<0.0001) for SND1, respectively, and 1.9, 1.0, 0.47, and 0.45, (p<0.0001) for AMACR, respectively. The intensity of SND1 immunoreactivity showed distinct correlation with Gleason score; more intense immunoreactivity being associated with higher specimen score (p=0.025). Expression of SND1 was also associated with high PSA but not with pathological T stage (p=0.012 and 0.95, respectively). By contrast, AMACR showed no relationship with any clinicopathological parameters including Gleason score, PSA level, and pathological T stage (p=0.65, 0.85 and 0.60, respectively). SND1 mRNA was also highly expressed in cancer cells but was negative to weak in noncancerous cells. The SND1 knocked down PC-3 cells were significantly suppressed in their growth by 47.3% as compared with negative control cells (p=0.030). CONCLUSIONS SND1 may have the potential for identification of the more aggressive and clinically significant prostate cancers. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e829 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hidetoshi Kuruma Tokyo, Japan More articles by this author Yuko Kamata Tokyo, Japan More articles by this author Hiroyuki Takahashi Tokyo, Japan More articles by this author Koji Igarashi Kanagawa, Japan More articles by this author Takahiro Kimura Tokyo, Japan More articles by this author Kenta Miki Tokyo, Japan More articles by this author Jun Miki Tokyo, Japan More articles by this author Hiroshi Sasaki Tokyo, Japan More articles by this author Norihiro Hayashi Tokyo, Japan More articles by this author Shin Egawa Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Human TRIB2 is a repressor of FOXO that contributes to the malignant phenotype of melanoma cells

FOXO transcription factors are evolutionarily conserved proteins that orchestrate gene expression programs known to control a variety of cellular processes such as cell cycle, apoptosis, DNA repair and protection from oxidative stress. As the abrogation of FOXO function is a key feature of many tumor cells, regulation of FOXO factors is receiving increasing attention in cancer research. In order to discover genes involved in the regulation of FOXO activity, we performed a large-scale RNA-mediated interference (RNAi) screen using cell-based reporter systems that monitor transcriptional activity and subcellular localization of FOXO. We identified genes previously implicated in phosphoinositide 3-kinase/Akt signaling events, which are known to be important for FOXO function. In addition, we discovered a previously unrecognized FOXO-repressor function of TRIB2, the mammalian homolog of the Drosophila gene tribbles. A cancer-profiling array revealed specific overexpression of TRIB2 in malignant melanoma, but not in other types of skin cancer. We provide experimental evidence that TRIB2 transcript levels correlate with the degree of cytoplasmic localization of FOXO3a. Moreover, we show that TRIB2 is important in the maintenance of the oncogenic properties of melanoma cells, as its silencing reduces cell proliferation, colony formation and wound healing. Tumor growth was also substantially reduced upon RNAi-mediated TRIB2 knockdown in an in vivo melanoma xenograft model. Our studies suggest that TRIB2 provides the melanoma cells with growth and survival advantages through the abrogation of FOXO function. Altogether, our results show the potential of large-scale cell-based RNAi screens to identify promising diagnostic markers and therapeutic targets.

WEE2 Is an Oocyte-Specific Meiosis Inhibitor in Rhesus Macaque Monkeys1

WEE1 homolog 2 (WEE2, also known as WEE1B) is a newly identified member of the WEE kinase family that is conserved from yeast to humans. The aim of the present study was to determine the spatiotemporal expression pattern and the function of WEE2 during oocyte maturation in a nonhuman primate species, the rhesus macaque. Among 11 macaque tissues examined, WEE2 transcript is predominantly expressed in the ovary and only weakly detectable in the testis. Within the ovary, WEE2 mRNA is exclusively localized in the oocyte and appears to accumulate during folliculogenesis, reaching the highest level in preovulatory follicles. Microinjection of a full-length WEE2-GFP (green fluorescent protein) fusion mRNA indicates a specific nuclear localization of WEE2 protein in both growing and fully grown germinal vesicle (GV)-intact oocytes. Taking the long double-stranded RNA-mediated RNA interference approach, we found that down-regulation of WEE2 led to meiotic resumption in a subset of GV oocytes even in the presence of a phosphodiesterase 3 inhibitor. On the other hand, overexpression of WEE2 delays the reentry of oocytes into meiosis in both mice and monkeys. These findings suggest that WEE2 is a conserved oocyte-specific meiosis inhibitor that functions downstream of cAMP in nonhuman primates.

Genome-wide RNA-mediated interference screen identifies miR-19 targets in Notch-induced T-cell acute lymphoblastic leukaemia

MicroRNAs (miRNAs) have emerged as novel cancer genes. In particular, the miR-17-92 cluster, containing six individual miRNAs, is highly expressed in haematopoietic cancers and promotes lymphomagenesis in vivo. Clinical use of these findings hinges on isolating the oncogenic activity within the 17-92 cluster and defining its relevant target genes. Here we show that miR-19 is sufficient to promote leukaemogenesis in Notch1-induced T-cell acute lymphoblastic leukaemia (T-ALL) in vivo. In concord with the pathogenic importance of this interaction in T-ALL, we report a novel translocation that targets the 17-92 cluster and coincides with a second rearrangement that activates Notch1. To identify the miR-19 targets responsible for its oncogenic action, we conducted a large-scale short hairpin RNA screen for genes whose knockdown can phenocopy miR-19. Strikingly, the results of this screen were enriched for miR-19 target genes, and include Bim (Bcl2L11), AMP-activated kinase (Prkaa1) and the phosphatases Pten and PP2A (Ppp2r5e). Hence, an unbiased, functional genomics approach reveals a coordinate clampdown on several regulators of phosphatidylinositol-3-OH kinase-related survival signals by the leukaemogenic miR-19.

Nanoparticle-Based Delivery System for Application of siRNA In Vivo

Small interfering RNAs (siRNAs) silence the expression of specific target genes by mediating RNA interference (RNAi) in mammalian cells. siRNAs have not only been widely used as a valuable tool for functional genomics research, but they also have demonstrated great potential in biomedical therapeutic applications for diseases caused by abnormal gene overexpression or mutation. One of the most important issues to overcome before full clinical application is the development of effective administration methods for siRNAs to the target tissue or cells in vivo, which is highly dependent on the delivery system. Currently, there are two major kinds of in vivo delivery systems: viral or nonviral. As one of the nonviral carrier systems, nanoparticles, combinations of liposomes and cationic polymer complexes, have exhibited improved in vivo stability, target specificity, and cell/tissue uptake and internalization of the encapsulated RNAi oligos, which result in more effective silencing with less cellular toxicity and immune stimulation. This review will discuss the latest advancements in nanoparticle-mediated RNAi delivery systems, including nano-materials, preparation, and characteristics. In conjunction, the clinical trial cases related to the nanoparticle-siRNA complexes will be highlighted. The safety issues of nanoparticles used in vivo will also be mentioned. Finally, this review will summarize the perspectives for future applications of nanoparticle-mediated RNAi delivery systems.

Centrosome Size Sets Mitotic Spindle Length in Caenorhabditis elegans Embryos

Just as the size of an organism is carefully controlled, the size of intracellular structures must also be regulated. The mitotic spindle is a supramolecular machine that generates the forces which separate sister chromatids during mitosis. Although spindles show little size variation between cells of the same type, spindle length can vary at least 10-fold between different species. Recent experiments on spindle length showed that in embryonic systems spindle length varied with blastomere size. Furthermore, a comparison between two Xenopus species showed that spindle length was dependent on some cytoplasmic factor. These data point toward mechanisms to scale spindle length with cell size. Centrosomes play an important role in organizing microtubules during spindle assembly. Here we use Caenorhabditis elegans to study the role of centrosomes in setting spindle length. We show that spindle length correlates with centrosome size through development and that a reduction of centrosome size by molecular perturbation reduces spindle length. By systematically analyzing centrosome proteins, we show that spindle length does not depend on microtubule density at centrosomes. Rather, our data suggest that centrosome size sets mitotic spindle length by controlling the length scale of a TPXL-1 gradient along spindle microtubules.

Integrin Stimulation Favors Uptake of Macromolecules by Cardiomyocytes In Vitro

Previously, our research group showed that integrin stimulation induces release of cardiac troponin I from viable neonatal rat ventricular cardiomyocytes (NRCMs), but would it also stimulate uptake of exogenous macromolecules? For this purpose, beating NRCMs were incubated without or with an RGD motif-containing peptide (GRGDS) to stimulate integrins in the presence of Texas Red-conjugated ovalbumin (OTR; 45 kDa) or dextran (DTR; 70 kDa). After incubation periods of 8, 16 and 24 hours endocytosis of red label was quantified by fluorescence microscopy. Uptake of OTR and DTR by NRCMs was intensified by GRGDS treatment (p for trend <0.001 and 0.019, respectively) and increased with duration of incubation (p<0.001 for both). The GRGDS-induced uptake of OTR by NRCMs correlated positively with OTR concentration (p<0.001). Experiments with pharmacological inhibitors of endocytosis indicated that in the absence of GRGDS, NRCMs take up OTR by the clathrin-mediated pathway of endocytosis while the GRGDS-dependent OTR uptake occurs by macropinocytosis. Cultures of NRCMs that were stretched cyclically showed ≈4-fold increased uptake of OTR compared to stationary NRCM cultures. Immunofluorescence microscopy revealed that the dysferlin-positive plasma membrane (PM) areas in beating GRGDS-treated NRCMs were ≈3-fold larger than in contracting NRCMs incubated with vehicle (p<0.001). However, in non-beating NRCMs exposure to GRGDS did not induce larger dysferlin-positive PM areas, nor did it stimulate uptake of OTR. After inhibition of dysferlin expression by short hairpin RNA-mediated RNA interference, OTR uptake by contracting NRCMs could no longer be stimulated via GRGDS treatment. We conclude that in NRCMs, stimulation of integrins by RGD motif-containing peptides or stretch cause uptake of labeled macromolecules. The latter process appears to depend on the contractile behavior of the NRCMs and on the PM repair protein dysferlin, probably because of its role in macropinocytosis.

Characterisation of Estrogen Responses in Breast Cancer Cells Highlights LRH-1 as a New Target for Breast Cancer Treatment.

Abstract Background: Liver receptor homolog-1 (LRH-1; NR5A2) is an orphan member of the Ftz-F1 family of nuclear receptors, which comprises four members (NR5A1-NR5A4). It is expressed in endodermal derived tissues and has been linked to a number of developmental, metabolic and proliferative processes. LRH-1 plays an important role in cholesterol and lipid homeostasis. Recent findings have shown that intestinal production of glucocorticoids is regulated by LRH-1, a deficiency of which sensitizes mice to colitis. Uncontrolled LRH-1 activity participates in the induction of intestinal tumours. LRH-1 protein expression has also been detected by immunohistochemistry in tumour cells of human mammary ductal carcinomas.Materials and Methods: Using a panel of breast cancer cell lines and human tissues we have studied the expression of LRH-1 together with another Ftz-F1 subfamily member steroidogenic factor 1 (SF-1) and the potent LRH-1 co-repressors small heterodimer partner (SHP) and DAX-1/Dax-1 (dosage-sensitive sex-reversal adrenal hypoplasia congenital critical region on the X chromosome 1). RNA ligase Mediated Rapid amplification of 5' complementary DNA ends (5'RLM-RACE) was performed in order to map the estrogen regulated transcription start sites for the LRH-1 gene. No natural ligand for LRH-1 has been described. Using fluorescence resonance energy transfer (FRET)-based assays a series of unusual bicyclic compounds have been defined as potent LRH-1 ligands. The effects of these compounds on breast cancer cell growth and gene expression has been investigated using SRB growth assays and quantitative RT-PCR.Results &amp; Discussion: LRH-1 expression was found to be highest in estrogen receptor positive cell lines and breast, colon,small intestine and liver tissue. Treatment with 17β-estradiol increased LRH-1 levels in estrogen receptor positive cell lines, while treatment with anti-estrogens Tamoxifen and ICI 182,780 (Fulvestrant) reduced LRH-1 levels. siRNA mediated RNA interference against LRH-1 significantly reduced the estrogen-dependent growth of estrogen receptor positive breast cancer cell lines. We have shown that LRH-1 regulates the expression of the LRH-1 target gene Inhibin-α (a member of the transforming growth factor-β superfamily) in breast cancer cell lines. 5' RACE identified two new variant forms of LRH-1, which we have named LRH-1 289 and LRH-1 325 respectively. These arise from a novel promoter region lying between exon 2 and exon 3. RT- PCR for the LRH-1 289 variant showed E2 regulation in MCF7 cells and was seen to account for the majority of LRH-1 expression in this line. SRB growth assays demonstrated that these bicyclical compounds stimulate the growth of estrogen receptor/LRH-1 positive breast cancer cell lines in a dose dependent manner. Luciferase assays have demonstrated a titratable increase in LRH-1 reporter activity following the addition of these compounds.Our studies show that LRH-1 is a potently estrogen regulated gene in breast cancer cells, which acts to regulate cell growth and gene expression. These results therefore help validate LRH-1 as a potential new target for breast cancer treatment Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4143.

RNAi Applications in Therapy Development for Neurodegenerative Disease

RNA-mediated interference (RNAi) is a powerful tool for experimental manipulation of gene expression and is widely used to investigate gene function both in vitro and in vivo. RNAi refers to an evolutionarily conserved cellular mechanism for sequence-specific post-transcriptional gene silencing, in which double-stranded RNAs promote selective degradation of homologous cellular mRNAs. Because RNAi-based techniques can be employed to reduce expression of specific genes, this approach holds great promise as a therapy for diverse diseases, including devastating neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases and amyotrophic lateral sclerosis (ALS). Importantly, in recent years RNAi has also emerged as a key tool in target identification and validation studies designed to complement traditional (i.e., small molecule-based) drug development strategies. These studies harness the power of RNAi-mediated reverse genetics to probe disease-associated pathways in both cell-based and animal models, and thus may provide critical data needed to focus drug development efforts around disease-relevant targets. This review highlights recent progress in the preclinical development of RNAi-based therapeutics for neurodegenerative disease and discusses the particular challenges that disorders of the central nervous system (CNS) pose for this approach. It further describes current applications of RNAi techniques for target identification and validation studies and underscores the importance of this methodology to developing treatments for neurological diseases.

Protein disulphide isomerase is required for signal peptide peptidase-mediated protein degradation

The human cytomegalovirus glycoprotein US2 induces dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol and targets them for proteasomal degradation. Signal peptide peptidase (SPP) has been shown to be integral for US2-induced dislocation of MHC class I heavy chains although its mechanism of action remains poorly understood. Here, we show that knockdown of protein disulphide isomerase (PDI) by RNA-mediated interference inhibited the degradation of MHC class I molecules catalysed by US2 but not by its functional homolog US11. Overexpression of the substrate-binding mutant of PDI, but not the catalytically inactive mutant, dominant-negatively inhibited US2-mediated dislocation of MHC class I molecules by preventing their release from US2. Furthermore, PDI associated with SPP independently of US2 and knockdown of PDI inhibited SPP-mediated degradation of CD3delta but not Derlin-1-dependent degradation of CFTR DeltaF508. Together, our data suggest that PDI is a component of the SPP-mediated ER-associated degradation machinery.

Downregulation of the Cell-Cycle Regulating Ubiquitin-Ligase APC/CCdh1 May Contribute to the Differentiation Block of AML1/Eto Positive AML.

Abstract 5045 IntroductionThe anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase regulating cell cycle progression by targeting various cell cycle regulators for proteasomal degradation. It is activated by the adaptor proteins Cdc20 in mitosis and by Cdh1 in late mitosis and G1/G0. Thereby, Cdh1 establishes a stable G1 phase enabling the cell to either exit the cell cycle and differentiate or to prepare for a new round of cell division. It has also been shown that Cdh1 plays a role in the differentiation of various cell types, such as neurons, myocytes, hepatocytes and lens epithelial cells. Methods and ResultsWe have examined the regulation of Cdh1 in several acute myeloid leukemia (AML) cell lines. We found that in the AML1/Eto positive leukemia cell lines SKNO-1 and Kasumi-1, Cdh1 protein and RNA levels are lower than in AML1/Eto negative cell lines KG-1 and HL-60. In addition, Cdh1 protein level in an AML1/Eto positive primary blast sample was lower than in AML1/Eto negative patient samples. The translocation t(8;21) is one of the most frequent chromosomal rearrangement in AML and results in an AML1/Eto fusion protein, which can act as a transcriptional repressor. Thus, our results are consistent with AML1/Eto mediated downregulation of Cdh1. To evaluate the potential role of APC/CCdh1 in myeloid differentiation, we established a stable Cdh1 knockdown (kd) in the AML1/Eto negative HL60 cell line with high Cdh1 expression by lentiviral vector mediated RNA interference. HL60 cells harbouring either a Cdh1 shRNA or a control shRNA against GFP were established simultaneously. We used PMA at concentrations of 0.5, 1, 2 and 50 nM to differentiate these cells into CD11b positive macrophage-like cells over 48h. Protein isolation and analysis of CD11b expression by flow cytometry were performed at 0, 6h, 12h, 24h and 48h to examine differentiation kinetics. Cdh1 and target proteins with a potential role in cell cycle arrest and differentiation, such as Skp2 (an activator of the SCF-ubiquitin ligase targeting p21 and p27) and ID2 (inhibitor of differentiation 2), were analyzed by Western blotting. We observed that kd of Cdh1 in HL60 cells resulted in 10% to 20% lower CD11b expression at any time, when PMA was used at concentrations 0, 0.5, 1nM over 48h. ID2 and Skp2 were stabilized in these Cdh1 kd cells compared to the control correlating with the less differentiated state. In addition, HL60 cells with a stable Skp2 kd showed a higher CD11b expression indicating a more differentiated status compared to the control. ConclusionThis is the first report that indicates a role for APC/CCdh1 in the differentiation of myeloid cells with SCFSkp2 being one of the relevant targets. Downregulation of Cdh1 may contribute to the differentiation block of AML1/Eto postive AML. DisclosuresNo relevant conflicts of interest to declare.

Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway

In flies, small silencing RNAs are sorted between Argonaute1 (Ago1), the central protein component of the microRNA (miRNA) pathway, and Argonaute2 (Ago2), which mediates RNA interference. Extensive double-stranded character-as is found in small interfering RNAs (siRNAs)-directs duplexes into Ago2, whereas central mismatches, like those found in miRNA/miRNA* duplexes, direct duplexes into Ago1. Central to this sorting decision is the affinity of the small RNA duplex for the Dcr-2/R2D2 heterodimer, which loads small RNAs into Ago2. Here, we show that while most Drosophila miRNAs are bound to Ago1, miRNA* strands accumulate bound to Ago2. Like siRNA loading, efficient loading of miRNA* strands in Ago2 favors duplexes with a paired central region and requires both Dcr-2 and R2D2. Those miRNA and miRNA* sequences bound to Ago2, like siRNAs diced in vivo from long double-stranded RNA, typically begin with cytidine, whereas Ago1-bound miRNA and miRNA* disproportionately begin with uridine. Consequently, some pre-miRNA generate two or more isoforms from the same side of the stem that differentially partition between Ago1 and Ago2. Our findings provide the first genome-wide test for the idea that Drosophila small RNAs are sorted between Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide.

RNAi for the Treatment of Prion Disease: A Window for Intervention in Neurodegeneration?

Effective treatment of neurodegenerative disease is one of the major challenges facing biomedical research. These disorders, which include Alzheimer's, Huntington's and Parkinson's diseases - as well as the rarer prion diseases - constitute an ever-increasing burden in the developed world, socially, medically and economically. The key barrier to effective therapy is that they present clinically when neuronal loss is advanced, and irreversible. Current treatments are almost all directed at modifying symptoms; few address underlying pathogenic mechanisms and are inevitably delivered too late to rescue dying neurons. In the field of prion diseases, however, insights into the molecular basis and the temporal evolution of prion neurotoxicity are increasing. Recent work in mice leads to new hope for the treatment of these disorders, and potentially for rescuing neurodegeneration more broadly. Using lentivirally mediated RNA interference (RNAi) against native prion protein (PrP), White et al report the first intervention resulting in neuronal rescue, prevention of symptoms and increased survival in mice with established prion disease. Central to the effectiveness of this strategy are both the target and the timing of the intervention: the treatment prevents the formation of the neurotoxic prion agent at a point when diseased neurons can still be saved from death. This review introduces the basic concepts of prion pathogenesis, emerging insights into mechanisms of prion neurotoxicity and the rationale for targeting endogenous prion protein (PrP) in prion therapeutics. It describes the discovery of a window of reversibility of early neuronal damage in prion disease and how together these advances led to the subsequent development of the strategy using RNAi based therapy for these disorders. It discusses the use and relevance of this approach more broadly in neurodegeneration.

Identification of LATS transcriptional targets in HeLa cells using whole human genome oligonucleotide microarray

Human LATS1 and LATS2) (LATS1/2) are tumor suppressors that have been shown to be mutated or downregulated in several human cancers including leukemia, lung, prostate and breast cancers. However, the precise mechanisms and the proteins modulated by LATS1/2 that are responsible for these events remain largely unknown. To elucidate potential signaling pathways, the current study investigated the expression profile in HeLa cells with reduced expression of LATS1/2. Using RNA-mediated interference, both LATS1 and LATS2 were substantially knocked-down, and accordingly, this lead to an increase in multiple phenotypes associated with tumor progression, including enhanced cell proliferation, resistance to drug-induced cell death, and increased cell migration. Using whole human genome Oligo (60-mer) arrays (Agilent), genes modulated by loss of LATS1/2 were identified and functionally grouped into categories including cell proliferation, cell death, cell adhesion and motility, as well as cell communication. Selected genes, including known tumor suppressor genes and oncogenes such as CDKN1A, WISP2, SLIT2, TP53INP1, BIRC4BP, SPRY2, SPRY4, SPRED1, FAT4, and CYR61 were confirmed by qRT-PCR to be significantly differentially expressed. Importantly, the collection of genes identified suggests that LATS1/2 function through diverse mechanisms and multiple signaling pathways including the Hippo signaling pathway, as well as the p53, Ras-ERK, or WNT networks, to inhibit tumor progression.

Knockdown of angiotensinogen by shRNA-mediated RNA interference inhibits human visceral preadipocytes differentiation

The immediate cause of obesity is the massive deposition of subcutaneous and visceral fat attributing to the continuous proliferation and differentiation of preadipocytes. The identification of the underlying molecular mechanisms of preadipocytes differentiation is urgent, and will have an important role in plastic and reconstructive surgical procedures. Two small hairpin RNA (shRNA)-mediated RNA interference plasmids have been constructed on the basis of the activity of H1 promoter-driven expression vector psiRNA-hH1neo to suppress the expression of angiotensinogen (AGT) in human preadipocytes-visceral (HPA-v). Subsequently, glycerol-3-phosphate dehydrogenase (G3PDH) activity and intracytoplasmic lipids content were detected during the process of HPA-v differentiation. Small hairpin RNA-expressing vectors have been successfully constructed to suppress the expression of AGT significantly. Both intracytoplasmic lipids content and G3PDH activity decreased to a certain extent compared with that in the control group in the whole process of HPA-v differentiation. Two shRNA-mediated AGT-targeting plasmids inhibited the process of HPA-v differentiation to a certain extent. However, the accumulation of intracytoplasmic lipids was not exclusively determined by the expression of AGT, and it may also be regulated by other factors. In conclusion, this study provided a method to inhibit the process of preadipocytes differentiation, and it may have a role in obesity treatment and adipose tissue engineering application.