siRNA Probes Research Articles

Determination of the interior pH of lipid nanoparticles using a pH-sensitive fluorescent dye-based DNA probe

Lipid nanoparticles (LNPs) containing ionizable cationic lipids are proven delivery systems for therapeutic nucleic acids, such as small interfering RNA (siRNA). It is important to understand the relationship between the interior pH of LNPs and the pH of the external environment to understand LNP formulation and function. Here, we developed a simple and rapid approach for determining the pH of the LNP core using a pH-sensitive fluorescent dye-based DNA probe. LNP siRNA systems containing pH-responsive DNA probes (LNP-siRNA&DNA) were generated by rapid mixing of lipids in ethanol and pH 4 aqueous buffer containing siRNA and DNA probes. We demonstrated that DNA probes were readily encapsulated in LNP systems and were sequestered into an environment at a high concentration as evidenced by an inter-probe FRET signal. It was shown that the pH of LNP encapsulated probes closely follows the pH increase or decrease of the external environment. This indicates that the clinically approved LNP RNA systems with similar lipid compositions (e.g., Onpattro and Comirnaty) are highly permeable to protons and that the pH of the interior environment closely mirrors the external environment. The pH-dependent response of the probe in LNPs was also confirmed under buffer conditions at various pHs. Furthermore, we showed that the pH-sensitive DNA probe can be incorporated into LNP systems at levels that allow the pH response to be monitored at a single LNP level using convex lens-induced confinement (CLiC) confocal microscopy. Direct visualization of the internal pH of single particles with the fluorescent DNA probe was achieved by CLiC for LNP-siRNA&DNA systems formulated under both high and normal ionic strength conditions.

Persistent COUP-TFII Expression Underlies the Myopathy and Impaired Muscle Regeneration Observed in Resistance to Thyroid Hormone-Alpha

Myopathic changes, including muscular dystrophy and weakness, are commonly described in hypothyroid and hyperthyroid patients. Thyroid hormone signaling, via activation of thyroid nuclear receptor alpha (THRA), plays an essential role in maintaining muscle mass, function, and regeneration. A mouse model of resistance to thyroid hormone carrying a frameshift mutation in the THRA gene (THRA-PV) is associated with accelerated skeletal muscle loss with aging and impaired regeneration after injury(1,2). We previously demonstrated that the expression of nuclear orphan receptor chicken ovalbumin upstream promoter-factor II (COUP-TFII, or Nr2f2) persists during myogenic differentiation in THRA-PV myoblasts and skeletal muscle of aged THRA- PV mice. COUP-TFII is known to regulate myogenesis negatively and has a role in Duchenne-like Muscular Dystrophies(3). COUP-TFII physically and functionally interacts with THRA in primary myoblasts isolated from WT and THRA-PV mice, as demonstrated via co-immunoprecipitation and chromatin-immunoprecipitation. We observed that satellite cells from THRA-PV mice display impaired myoblast proliferation and in vitro myogenic differentiation compared to WT cells. However, the silencing of COUP-TFII expression using siRNA probes restores in vitro myogenic potential of THRA-PV myoblasts and shifts the mRNA expression profile closer to WT myoblasts, with a higher proliferation of myoblasts and a higher number of fully differentiated myotubes after 5 days of myogenic induction. Moreover, RNAseq analysis on myoblasts from THRA-PV mice after COUP-TFII knockdown shows that COUP-TFII silencing reverses the transcriptomic profile of THRA-PV myoblasts and results in reactivation of pathways involved in muscle function and extracellular matrix remodeling/deposition. These findings indicate that the persistent COUP-TFII expression in THRA-PV mice is responsible for the abnormal muscle phenotype. In conclusion, COUP-TFII and THRA cooperate during murine post-natal myogenesis, and COUP-TFII is critical for the accelerated skeletal muscle loss with aging and impaired muscle regeneration after injury in THRA-PV mice. These studies can help increase our knowledge of the mechanisms involved in thyroid hormone signaling during skeletal muscle regeneration, ultimately increasing the possibility of designing more specific treatments for patients with thyroid hormone-induced myopathies.

Caspase-Activated Oligonucleotide Probe.

Light-activated ("caged") oligonucleotides provide a strategy for modulating the activity of antisense oligos, siRNA, miRNA, aptamers, DNAzymes, and mRNA-capturing probes with high spatiotemporal resolution. However, the near-UV and visible wavelengths that promote these bond-breaking reactions poorly penetrate living tissue, which limits some biological applications. To address this issue, we describe the first example of a protease-activated oligonucleotide probe, capable of reporting on caspase-3 during cellular apoptosis. The 2'-F RNA-peptide substrate-peptide nucleic acid (PNA) hairpin structure was generated in 30% yield in a single bioconjugation step.

OR01-03 Thyroid Receptor Alpha Interacts with COUP-TFII in the Regulation of Postnatal Skeletal Muscle Regeneration

Myopathic changes, including muscular dystrophy and weakness, are commonly described in hypothyroid and hyperthyroid patients. Thyroid hormone signaling, via activation of thyroid nuclear receptors (TRs), plays an essential role in the maintenance of muscle mass, function, and regeneration. TRs are ligand-inducible transcription factors expressed in almost all tissues, including skeletal muscle. In a mouse model of Resistance to Thyroid Hormone carrying a frame-shift mutation in the TRα gene (TRα1PV)1,2 we observed skeletal muscle loss with aging and impaired skeletal muscle regeneration after injury. We recently described that TRα interacts with the nuclear orphan receptor Chicken Ovalbumin Upstream Promoter-factor II (COUP-TFII, or NR2F2), which is known to regulate myogenesis negatively and has a role in Duchenne-like Muscular Dystrophies3. We showed that COUP-TFII expression declines with age in WT mice, while the skeletal muscle of TRα1PV mice shows a sustained significantly higher expression of COUP-TFII. Our findings suggest that the TRα/COUP-TFII interaction might mediate the impaired skeletal muscle phenotype observed in TRα1PV mice. To better characterize this interaction, we isolated SC from 10 months old WT and TRα1PV mice and cultured them in vitro using novel methods established within our lab. Using siRNA probes, we next silenced COUP-TFII and characterized the cells via RNA-seq analysis. In vitro, we assessed myoblast differentiation and proliferation using differentiation assays and EdU incorporation. We observed that satellite cells from TRα1PV mice display impaired myoblast proliferation and in vitro myogenic differentiation compared to WT SCs. However, when COUP-TFII was silenced, the myogenic potential of TRα1PV satellite cells was restored, with a higher proliferation of myoblasts and a higher number of fully differentiated myotubes after 4 days of myogenic induction. RNAseq analysis on satellite cells from TRα1PV mice after COUP-TFII knockdown showed upregulation of genes involved in the myogenic pathway, such as Myod1 and Pax7, and of genes in the thyroid hormone signaling, such as Dio2. Ingenuity Pathway Analysis further showed activation of pathways regarding cell growth, differentiation, matrix remodeling along with muscle function, muscle contractility, and muscle contraction. These in vitro results suggest that by silencing COUP-TFII we promote the myogenic pathway and may further rescue the impaired phenotype of TRα1PV mice. These studies can help increase our knowledge of the mechanisms involved in thyroid hormone signaling in skeletal muscle regeneration, which will ultimately increase the possibility of designing more specific treatments for patients with thyroid hormone-induced myopathies.

Downregulation of the Arg/N-degron Pathway Sensitizes Cancer Cells to Chemotherapy In Vivo

The N-degron pathway is an emerging target for anti-tumor therapies, because of its capacity to positively regulate many hallmarks of cancer, including angiogenesis, cell proliferation, motility, and survival. Thus, inhibition of the N-degron pathway offers the potential to be a highly effective anti-cancer treatment. With the use of a small interfering RNA (siRNA)-mediated approach for selective downregulation of the four Arg/N-degron-dependent ubiquitin ligases, UBR1, UBR2, UBR4, and UBR5, we demonstrated decreased cell migration and proliferation and increased spontaneous apoptosis in cancer cells. Chronic treatment with lipid nanoparticles (LNPs) loaded with siRNA in mice efficiently downregulates the expression of UBR-ubiquitin ligases in the liver without any significant toxic effects but engages the immune system and causes inflammation. However, when used in a lower dose, in combination with a chemotherapeutic drug, downregulation of the Arg/N-degron pathway E3 ligases successfully reduced tumor load by decreasing proliferation and increasing apoptosis in amouse model of hepatocellular carcinoma, while avoiding theinflammatory response. Our study demonstrates that UBR-ubiquitin ligases of the Arg/N-degron pathway are promising targets for the development of improved therapies for many cancer types.

Myofibril Assembly in Cultured Mouse Neonatal Cardiomyocytes.

The formation of myofibrils was analyzed in neonatal mouse cardiomyocytes grown in culture and stained with fluorescent antibodies directed against myofibrillar proteins. The cardiomyocyte cultures also were exposed to siRNA probes to test the role of nonmuscle myosin IIB expression in the formation of myofibrils. In culture, new myofibrils formed in the spreading cell margins surrounding contractile myofibrils previously assembled in utero. Observations indicated that assembly of mature myofibrils occurred in three-stages, as previously reported in cultured mouse skeletal muscle. Premyofibrils, characterized by minisarcomeres with nonmuscle myosin IIB and muscle-specific alpha-actinin bound to actin filaments, formed in the first stage; followed by nascent myofibrils, the second stage when muscle myosin II and titin were first detected. In the mature myofibril stage muscle myosin II filaments aligned in periodic A-Bands; late assembling proteins, including myomesin and telethonin, were integrated in the sarcomeres, and nonmuscle IIB was absent from the sarcomeres. Treatment of the cultured neonatal cardiomyocytes with gene-specific siRNAs for nonmuscle myosin IIB, led to a marked decrease in the formation of premyofibrils, and subsequently of mature myofibrils. Anat Rec, 301:2067-2079, 2018. © 2018 Wiley Periodicals, Inc.

Dissipative particle dynamic simulation on the assembly and release of siRNA/polymer/gold nanoparticles based polyplex

Dissipative particle dynamics simulation is used to reveal the loading/release of small interfering RNA (siRNA) in pH‐sensitive polymers/gold nanoparticles (AuNPs) polyplex. The conformation dynamics of these polyplex at various Au/siRNA mass ratios, the original AuNPs sizes, polymer types, and pH values are simulated and compared to experimental results. At neutral conditions (pH = 7.4), spherical micelles with a multilayer structure are formed in siRNA/polyethyleneimine/cis‐aconitic anhydride functionalized poly(allylamine)/polyethyleneimine/11‐mercaptoundecanoic acid‐gold nanoparticle (siRNA/PEI/PAH‐Cit/PEI/MUA‐AuNP) polyplex. Large polyplex are obtained with high Au/siRNA mass ratio and/or small original AuNPs size. The release dynamics of siRNA from AuNPs‐polyplex systems were also simulated in the intracellular environment (pH = 5.0). A swelling‐demicellization‐releasing mechanism is followed while the release of siRNA is found much faster for polyplex involving charge‐reversal PAH‐Cit. These findings are qualitatively consistent with the experimental results and may provide valuable guidance in later design and optimization of delivery carriers for siRNA or other molecule probes. © 2017 American Institute of Chemical Engineers AIChE J, 64: 810–821, 2018

Size Specific Transfection to Mammalian Cells by Micropillar Array Electroporation.

Electroporation serves as a promising non-viral gene delivery approach, while its current configuration carries several drawbacks associated with high-voltage electrical pulses and heterogeneous treatment on individual cells. Here we developed a new micropillar array electroporation (MAE) platform to advance the electroporation-based delivery of DNA and RNA probes into mammalian cells. By introducing well-patterned micropillar array texture on the electrode surface, the number of pillars each cell faces varies with its plasma membrane surface area, despite their large population and random locations. In this way, cell size specific electroporation is conveniently carried out, contributing to a 2.5~3 fold increase on plasmid DNA transfection and an additional 10–55% transgene knockdown with siRNA probes, respectively. The delivery efficiency varies with the number and size of micropillars as well as their pattern density. As MAE works like many single cell electroporation are carried out in parallel, the electrophysiology response of individual cells is representative, which has potentials to facilitate the tedious, cell-specific protocol screening process in current bulk electroporation (i.e., electroporation to a large population of cells). Its success might promote the wide adoption of electroporation as a safe and effective non-viral gene delivery approach needed in many biological research and clinical treatments.

Deciphering the true antiproliferative target of an MK2 activation inhibitor in glioblastoma.

There has been much interest in developing inhibitors of the checkpoint kinases Chk1/2 to augment the effects of DNA-damaging agents for chemotherapy.1 In addition to Chk1/2, Wee1 and MAPK-activated protein kinase-2 (MK2) have emerged as additional key regulators of cell-cycle checkpoints.2 Evidence has accumulated that implicates MK2 as a target for chemo-sensitization in both p53-proficient and deficient tumors.3 MK2 is an attractive target for cancer treatment as MK2 inhibition has the potential to regulate the cell-cycle effects of the p38–MAPK pathway without inhibition of poly-functional p38 itself, which regulates many cellular signaling networks. MK2 inhibition in the absence of synergistic chemotherapy had not been investigated for its inherent cytotoxicity, and Munoz published a study aimed to fill this gap in our knowledge of MK2.4 For their MK2 cytotoxicity study in glioblastoma cells, Munoz et al. combined the use of siRNA and chemical probes. Three MK2 inhibitors were chosen for their distinct structures and mechanism of action (Figure 1a). CMPD1 is a non-ATP competitive inhibitor, which prevents phosphorylation and activation of MK2 via binding to p38–MAPK (Ki 330 nM).5 CMPD1 is not simply a non-selective p38 inhibitor as it shows no inhibition of the phosphorylation of two other p38 substrates, ATF2 and MBP. MK2i is a classical ATP-competitive kinase inhibitor, which inhibits MK2 (IC50 126 nM).2 PF-3644022 is a more potent ATP-competitive MK2 inhibitor (Ki 5 nM).6 Although MK2i and PF-3644022 are both ATP-competitive MK2 inhibitors, they differ significantly in potency and are structurally orthogonal.7

Silencing of xylose isomerase and cellulose synthase by siRNA inhibits encystation in Acanthamoeba castellanii

A key challenge in the successful treatment of Acanthamoeba infections is its ability to transform into a dormant cyst form that is resistant to physiological conditions and pharmacological therapies, resulting in recurrent infections. The carbohydrate linkage analysis of cyst walls of Acanthamoeba castellanii showed variously linked sugar residues, including xylofuranose/xylopyranose, glucopyranose, mannopyranose, and galactopyranose. Here, it is shown that exogenous xylose significantly reduced A. castellanii differentiation in encystation assays (P < 0.05 using paired t test, one-tailed distribution). Using small interfering RNA (siRNA) probes against xylose isomerase and cellulose synthase, as well as specific inhibitors, the findings revealed that xylose isomerase and cellulose synthase activities are crucial in the differentiation of A. castellanii. Inhibition of both enzymes using siRNA against xylose isomerase and cellulose synthase but not scrambled siRNA attenuated A. castellanii metamorphosis, as demonstrated by the arrest of encystation of A. castellanii. Neither inhibitor nor siRNA probes had any effect on the viability and extracellular proteolytic activities of A. castellanii.

Intra-tympanic delivery of short interfering RNA into the adult mouse cochlea

Trans-tympanic injection into the middle ear has long been the standard for local delivery of compounds in experimental studies. Here we demonstrate the advantages of the novel method of intra-tympanic injection through the otic bone for the delivery of compounds or siRNA into the adult mouse cochlea. First, a fluorescently-conjugated scrambled siRNA probe was applied via intra-tympanic injection into the middle ear cavity and was detected in sensory hair cells and nerve fibers as early as 6 h after the injection. The fluorescent probe was also detected in other cells of the organ of Corti, the lateral wall, and in spiral ganglion cells 48 h after the injection. Furthermore, intra-tympanic delivery of Nox3 siRNA successfully reduced immunofluorescence associated with Nox3 in outer hair cells 72 h after injection by 20%. Drug or siRNA delivery via intra-tympanic injection does not compromise the tympanic membrane or interfere with noise-induced hearing loss, while trans-tympanic injections significantly altered the cochlear response to noise exposure. In summary, intra-tympanic injection through the otic bone into the middle ear cavity provides a promising approach for delivery of compounds or siRNA to cochlear hair cells of adult mice, relevant for the study of mechanisms underlying inner ear insults and, specifically, noise-induced hearing loss.

Depletion of the TP53 Family Member p73 Sensitizes Cancer Cells towards Cytarabine.

AbstractAbstract 2461 Background:Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. 50–70 % of adults with AML achieve complete remission with induction of chemotherapy - only 20–30 % of patients enjoy long-term disease-free survival. Many AML-patients still die of their disease, most frequently because of drug resistance. We intended to study drug resistance in cancer cells by using a synthetic lethal RNAi screen with the S phase targeting drug cytarabine (Ara-C). Our goal was the identification of siRNAs that overcome drug resistance triggered by cytarabine. Methods:For the chemosensitizer RNAi screen we used lipid-based reverse transfection for transient siRNA-mediated gene silencing in the human osteosarcoma cell line (U2OS). The human osteosarcoma cell line is a good model to study cytarabine resistance: the cells are highly refractory to Ara-C treatment, while activating DNA damage response (indicated by activation of different DNA checkpoint markers (phospho-Chk1, phospho-p53, p21)). Gene silencing was done by using a custom library comprising 437 siRNA probes specific for DNA repair and DNA damage response genes (consisting of a pool of four different siRNA-sequences for one gene). Synthetic lethality of siRNAs with a sub lethal dose of cytarabine (IC20) was measured after 72 hours of incubation with a luminescence-based cell viability assay. Hits were identified after z-score normalization of each plate and by evaluating the cytarabine-sensitivity of siRNAs reducing cell viability in combination with Ara-C vs. cell viability of untreated siRNA transfected cells. Screens were done in duplicate and high confidence hits (with z-scores greater than 2 STD) were validated. For secondary validation screens and further validation experiments we used two different single siRNA-sequences for each gene. Results:We reproducible identified and validated candidate genes which sensitize cells to cytarabine-treatment when silenced. Our top hits p73 and components of ubiquitine ligase complexes have been implicated in drug resistance and some are also “druggable”. The positively screened p73, a member of the p53 tumor suppressor protein family, is frequently overexpressed in cancer patients and correlates with increased tumor aggressiveness and therapy resistance. Apoptosis of p73-depleted U2OS cells is dramatically increased in the presence of cytarabine (> 50 %) compared to untreated p73-depleted cancer cells. We provide data that p73 and other screen hits enhance the PCNA-coupled post-replicative DNA repair pathway as the basis for cytarabine resistance. Analyzing the genome-wide gene expression profiles of 286 AML-patients with different karyotypes showed that our top screen hits in human osteosarcoma cells have a prognostic impact in AML-patients. Kaplan-Meier survival analysis showed that AML-patients with high-level expression of p73 displayed a significantly reduced overall survival compared to p73 low-expressing AML-patients. Together, these data may lead to a more individualized AML therapy, resulting in better treatment outcome. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Development of siRNA-probes for studying intracellular trafficking of siRNA nanoparticles.

One important barrier facing the delivery of short interfering RNAs (siRNAs) via synthetic nanoparticles is the rate of nanoparticle disassembly. However, our ability to optimize the release kinetics of siRNAs from nanoparticles for maximum efficacy is limited by the lack of methods to track their intracellular disassembly. Towards this end, we describe the design of two different siRNA-based fluorescent probes whose fluorescence emission changes in response to the assembly state of the nanoparticle. The first probe design involves a redox-sensitive fluorescence-quenched probe that fluoresces only when the nanoparticle is disassembled in a reductive environment. The second probe design is based on a FRET-labeled siRNA pair that fluoresces due to the proximity of the siRNA pair when the nanoparticle is intact. In both approaches, the delivery vehicle need not be labeled. The utility of these probes was investigated with a lipidoid nanoparticle (LNP) as proof-of-concept in both extracellular and intracellular environments. Fluorescence kinetic data from both probes were fit to a two-phase release and decay curve and subsequently quantified to give intracellular disassembly rate constants. Quantitative analysis revealed that the rate constant of siRNA release measured via the fluorescence-quenched probe was almost identical to the rate constant for nanoparticle disassembly measured via the FRET-labeled probes. Furthermore, these probes were utilized to determine subcellular localization of LNPs with the use of automated high-resolution microscopy as they undergo dissociation. Interestingly, this work shows that large amounts of siRNA remain inside vesicular compartments. Altogether, we have developed new siRNA probes that can be utilized with multiple nanocarriers for quantitative and qualitative analysis of nanoparticle dissociation that may serve as a design tool for future delivery systems.

FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA Nanocomplexes

The assembly, stability, and timely disassembly of short interfering RNA (siRNA) nanocomplexes have the potential to affect the efficiency of siRNA delivery and gene silencing. As such, the design of new probes that can measure these properties without significantly perturbing the nanocomplexes or their environment may facilitate the study and further development of new siRNA nanocomplexes. Herein, we study Förster resonance energy transfer (FRET)-labeled siRNA probes that can track the assembly, stability, and disassembly of siRNA nanocomplexes in different environments. The probe is composed of two identical siRNAs, each labeled with a fluorophore. Upon nanocomplex formation, the siRNA-bound fluorophores become locally aggregated within the nanocomplex and undergo FRET. A key advantage of this technique is that the delivery vehicle (DV) need not be labeled, thus enabling the characterization of a large variety of nanocarriers, some of which may be difficult or even impossible to label. We demonstrate proof-of-concept by measuring the assembly of various DVs with siRNAs and show good agreement with gel electrophoresis experiments. As a consequence of not having to label the DV, we are able to determine nanocomplex biophysical parameters such as the extracellular apparent dissociation constants (K(D)) and intracellular disassembly half-life for several in-house and proprietary commercial DVs. Furthermore, the lack of DV modification allows for a true direct comparison between DVs as well as correlation between their biophysical properties and gene silencing.

Maspin is a deoxycholate-inducible, anti-apoptotic stress-response protein differentially expressed during colon carcinogenesis

Increased maspin expression in the colon is related to colon cancer risk and patient survival. Maspin is induced by the hydrophobic bile acid, deoxycholate (DOC), which is an endogenous carcinogen and inducer of oxidative stress and DNA damage in the colon. Persistent exposure of colon epithelial cells, in vitro, to high physiologic levels of DOC results in increased constitutive levels of maspin protein expression associated with the development of apoptosis resistance. When an apoptosis-resistant colon epithelial cell line (HCT-116RC) developed in the authors’ laboratory was treated with a maspin-specific siRNA probe, there was a statistically significant increase in apoptosis compared to treatment with an siRNA control probe. These results indicate, for the first time, that maspin is an anti-apoptotic protein in the colon. Immunohistochemical evaluation of maspin expression in human colonic epithelial cells during sporadic colon carcinogenesis (131 human tissues evaluated) indicated a statistically significant increase in maspin protein expression beginning at the polyp stage of carcinogenesis. There was no statistically significant difference in maspin expression between hyperplastic/adenomatous polyps and colonic adenocarcinomas. The absence of “field defects” in the non-neoplastic colonic mucosa of patients with colonic neoplasia indicates that maspin may drive the growth of tumors, in part, through its anti-apoptotic function.

Inducible Silencing of Junctophilins in Skeletal Muscle Leads to Reversible Remodeling of the Triad Junction Structure and Compromised Store-Operated Calcium Entry

Junctophilins (JPs) play an essential role in muscle excitation-contraction (E-C) coupling by contributing to the formation of junctional membrane complexes (JMCs). However, the lethality associated with germ-line ablation of either JP1 or JP2 prevents physiological evaluation of their function in the maintenance of calcium homeostasis in adult muscle fibers. To investigate the physiological role of JP genes, we developed a novel transgenic system for tissue-specific and inducible control of gene expression in mouse models. This system employs a Tet-response CMV promoter that controls expression of a small-hairpin (sh) RNA against JP1 and JP2, which is non-functional until an interrupting reporter gene cassette is excised by the Cre recombinase. Insertion of the natural Dicer and Drosha-RNAse processing sites within the shRNA sequence allows for generation of specific siRNA probe for efficient knockdown of JP1 and JP2. Under the tight control of doxycycline (Dox), tissue- or lineage-specific expression of siRNA is achieved by the use of inducer mice that expresss Cre in a given tissue. Transgenic mice with muscle-specific expression of shRNA against JP showed no apparent change of JP expression before treatment with Dox; inducible and reversible knockdown of JP in skeletal muscle was achieved through feeding of animals with diet containing Dox. Dox-induced reduction of JP expression led to abnormal membrane structure and compromised store-operated Ca2+ entry in adult muscle fibers, consistent with JP's essential role in muscle development and function. This transgenic system can be applicable for inducible and reversible knockdown of different genes and in different tissues, as well as for control of transgene overexpression in an inducible and tissue-specific manner, thus providing a versatile system for eluciding the physiological gene function using viable animal models.

SiRNA Delivery to CNS Cells using a Membrane Translocation Peptide

RNA interference (RNAi) is a sequence-specific gene silencing technique that has been applied to multiple pathological conditions. In this report, we describe the generation and in vitro characterization of an RNAi-based fluorescent probe for use as a therapeutic in the setting of ischemic stroke. Probe delivery to bEnd.3 brain endothelial cells and primary cortical neurons and astrocytes was promoted by incorporating small interfering RNA (siRNA) into complexes with fluorescently labeled myristoylated polyarginine peptides. The resulting probe was partially protected from serum nuclease degradation and was efficiently internalized by cells as confirmed by flow cytometry and confocal microscopy. In addition, application of the siRNA probe directed against c-Src, a protein implicated in stroke pathology, led to statistically significant reduction of endogenous c-src mRNA levels in all cell types tested. Results demonstrate the proof-of-principle that functionalized peptide--siRNA probes can be used as potential tools for dual imaging and therapeutic applications.

Abstract 4063: Estrogen receptor alpha functions as an RNA binding protein to regulate GATA3 protein levels: a novel role for ER

Abstract The role of estrogen receptor alpha (ER) as a transcription factor has been well established. ER transcriptionally activates genes by binding to DNA directly within the gene promoter region or by binding to DNA indirectly through protein-protein dependent mechanisms. Many ER-responsive genes that are activated through ER-mediated transcriptional activation have been identified, including GATA3. While the role of GATA3 in breast cancer still remains largely unknown, numerous reports have shown that ER and GATA3 are coordinately expressed in breast cancer and that ER may play a role in regulating GATA3 expression. A recent report by Eeckhoute et al (2007) has shown that ER transcriptionally activated GATA3 expression in T47D breast cancer cells. Using MCF7 cells, we have recently identified a novel role for ER as an RNA binding protein that binds to GATA3 mRNA. Interestingly, silencing of ER using siRNA probes slightly increased GATA3 mRNA levels, however GATA3 protein levels decreased significantly. This decrease in protein was not due to improper export of GATA3 mRNA from the nucleus to the cytoplasm where protein translation occurs, as GATA3 mRNA expression was equivalent in both the nuclear and cytoplasmic compartments. This suggested that the decrease in protein expression was not due to impaired GATA3 mRNA transport but could be due to impaired protein translation. Since silencing of ER had dramatically different effects on RNA and protein levels, this data suggested that ER protein may function as an RNA binding protein that binds to the GATA3 mRNA to regulate its protein translation. Using biotin pulldown techniques, we found that ER does function as an RNA binding protein by binding directly to a site within a 125 nucleotide region of the GATA3 3‘UTR. This role of ER as an RNA binding protein is consistent with the data of Lanz et al (1999, 2000) that demonstrated that ER protein can bind to an RNA transcript, the steroid receptor co-activator, SRA. When bound to SRA, however, ER did not alter the RNA levels or protein expression of SRA but did result in increased activation of ER. Therefore, our data is the first to identify a novel role for ER as a RNA binding protein that can regulate the protein expression of the RNA transcript to which it is bound. Altering such ER-RNA interactions could potentially provide a new therapeutic avenue for the treatment of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4063.

Glucose stimulates cholesterol 7α-hydroxylase gene transcription in human hepatocytes

Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4alpha (HNF4alpha) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4alpha to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.

Myeloperoxidase serves as a redox switch that regulates apoptosis in epithelial ovarian cancer

Objectives Resistance to apoptosis is a key feature of cancer cells and is believed to be regulated by nitrosonium ion (NO +)-induced S-nitrosylation of key enzymes. Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS), is utilized by MPO to generated NO +. We sought to investigate the expression of myeloperoxidase (MPO) and iNOS in epithelial ovarian cancer (EOC) and determine their effect on S-nitrosylation of caspase-3 and its activity as well as apoptosis. Methods MPO and iNOS expression were determined using immunofluorescence in SKOV-3 and MDAH-2774 and EOC tissue sections. S-nitrosylation of caspase-3 and its activity, levels of MPO and iNOS, as well as apoptosis, were evaluated in the EOC cells before and after silencing MPO or iNOS genes with specific siRNA probes utilizing real-time RT–PCR, ELISA, and TUNEL assays. Results MPO and iNOS are expressed in EOC cell lines and in over 60% of invasive EOC cases with no expression in normal ovarian epithelium. Indeed, silencing of MPO or iNOS gene expression resulted in decreased S-nitrosylation of caspase-3, increased caspase-3 activity, and increased apoptosis but with a more significant effect when silencing MPO. Conclusion MPO and iNOS are colocalized to the same cells in EOC but not in the normal ovarian epithelium. Silencing of either MPO or iNOS significantly induced apoptosis, highlighting their role as a redox switch that regulates apoptosis in EOC. Understanding the mechanisms by which MPO functions as a redox switch in regulating apoptosis in EOC may lead to future diagnostic tools and therapeutic interventions.