Nonspecific Oligonucleotide Research Articles

Magnetic Nanoscalpel for the Effective Treatment of Ascites Tumors

One of the promising novel methods for radical tumor resection at a single-cell level is magneto-mechanical microsurgery (MMM) with magnetic nano- or microdisks modified with cancer-recognizing molecules. A low-frequency alternating magnetic field (AMF) remotely drives and controls the procedure. Here, we present characterization and application of magnetic nanodisks (MNDs) as a surgical instrument ("smart nanoscalpel") at a single-cell level. MNDs with a quasi-dipole three-layer structure (Au/Ni/Au) and DNA aptamer AS42 (AS42-MNDs) on the surface converted magnetic moment into mechanical and destroyed tumor cells. The effectiveness of MMM was analyzed on Ehrlich ascites carcinoma (EAC) cells in vitro and in vivo using sine and square-shaped AMF with frequencies from 1 to 50 Hz with 0.1 to 1 duty-cycle parameters. MMM with the "Nanoscalpel" in a sine-shaped 20 Hz AMF, a rectangular-shaped 10 Hz AMF, and a 0.5 duty cycle was the most effective. A sine-shaped field caused apoptosis, whereas a rectangular-shaped field caused necrosis. Four sessions of MMM with AS42-MNDs significantly reduced the number of cells in the tumor. In contrast, ascites tumors continued to grow in groups of mice and mice treated with MNDs with nonspecific oligonucleotide NO-MND. Thus, applying a "smart nanoscalpel" is practical for the microsurgery of malignant neoplasms.

Biophysical characterization of the DNA binding motif of human phospholipid scramblase 1.

Human phospholipid scramblase 1 (hPLSCR1) is a 37kDa multi-compartmental protein, which was initially identified as a Ca2+-dependent phospholipid translocator upon localizing to the plasma membrane. However, under certain physiological conditions, hPLSCR1 is localized to the nucleus where it interacts with the IP3R1 promoter (IP3R1P) and regulates its expression. In this study, the DNA binding properties of hPLSCR1 and ∆100-hPLSCR1 (N-terminal 100 amino acids deleted from hPLSCR1) were investigated by using a synthetic IP3R1P oligonucleotide and nonspecific scrambled-sequence oligonucleotides. Our results revealed that hPLSCR1 and ∆100-hPLSCR1 were bound to IP3R1P oligos in a 1:1 stoichiometry. In addition, ∆160-hPLSCR1 could not bind to IP3R1P oligonucleotide suggesting that the proposed DNA binding motif is the actual DNA binding motif. Specific binding of hPLSCR1 and ∆100-hPLSCR1 to IP3R1P oligos was demonstrated by fluorescence anisotropy assay. ITC analysis revealed that hPLSCR1 binds to IP3R1P oligos with Kd = 42.91 ± 0.23nM. Binding of IP3R1P oligos induces β-sheet formation in hPLSCR1 and increases the thermal stability of hPLSCR1 and ∆100-hPLSCR1. Binding of IP3R1P oligos to hPLSCR1 altered the B-form of the DNA, which was not observed with ∆100-hPLSCR1. Results from this study suggest that (i) ∆100-hPLSCR1 possesses a minimal DNA binding region and (ii) structural alterations of IP3R1P oligo by hPLSCR1 require proline-rich N-terminal region.

Adeno-Associated Virus D-Sequence-Mediated Suppression of Expression of a Human Major Histocompatibility Class II Gene: Implications in the Development of Adeno-Associated Virus Vectors for Modulating Humoral Immune Response.

A 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.

Interactions Between Hemin-Binding DNA Aptamers and Hemin–Graphene Nanosheets: Reduced Affinity but Unperturbed Catalytic Activity

We demonstrate herein that hemin–graphene hybrid nanosheets (H–GNs) can differentiate hemin-binding DNA aptamers (i.e., guanine-rich DNA sequences that form G-quadruplex structures upon binding hemin) and nonspecific single-stranded oligonucleotides colorimetrically. By exploiting the tendency of the H–GNS to aggregate under high ionic strength and their inherent catalytic activities, excess H–GNS were removed and the amount of H–GNs remaining in the supernatant was quantified. Subsequently, the binding affinity of hemin-binding DNA aptamers to the H–GNS was determined upon titration with the DNA. With the conformity of the experimental data to the Langmuir adsorption isotherm, hemin is hypothesized to be sandwiched between the G-quadruplex DNA and graphene forming a ternary complex as a result of the specific binding of the aptamer with the H–GNS. The binding affinity of the aptamer is not as strong as that of hemin/G-quadruplex in solution, which is attributed to electronic effects imparted by the graphene surface. The catalytic activity of the resulting complex, however, was confirmed to be higher than that of H–GNs alone.

A highly sensitive miR-195 nanobiosensor for early detection of Parkinson’s disease

Early detection of Parkinson’s disease (PD), as a dangerous neurodegenerative disease, is a key factor in the therapy or prevention of further development of this disease. We developed an electrochemical nanobiosensor for early detection of PD based on the quantification of circulating biomarker, miR-195. Exfoliated graphene oxide (EGO) and gold nanowires (GNWs) were used to modify the surface of screen-printed carbon electrode. A single-strand thiolated probe was designed for specific hybridization with target miRNA (miR-195), and doxorubicin was used as an electrochemical indicator for differential pulse voltammetry measurements. The results of scanning electron microscope imaging and cyclic voltammetry experiments confirmed the accuracy of the working electrode modification steps. The results of analytical performance nanobiosensor showed a high sensitivity of the biosensing with 2.9 femtomolar detection limit and dynamic range of 10.0–900.0 femtomolar. In addition, good selectivity for target miRNA over non-specific oligonucleotides (one and three base replacement in target miRNA, and non-complementary) was achieved. The results of real human serum analysis did not show any interference in the function of the biosensor. Based on the results, the miR-195 electrochemical nanobiosensor could be suggested for clinicians in the medical diagnosis of PD.

Reversal of MicroRNA Dysregulation in an Animal Model of Pulmonary Hypertension.

BackgroundAnimals models have played an important role in enhancing our understanding of the pathogenesis of pulmonary arterial hypertension (PAH). Dysregulation of the profile of microRNAs (miRNAs) has been demonstrated in human tissues from PAH patients and in animal models. In this study, we measured miRNA levels in the monocrotaline (MCT) rat model of PAH and examined whether blocking a specific dysregulated miRNA not previously reported in this model, attenuated PAH. We also evaluated changes in miRNA expression in lung specimens from MCT PAH rats overexpressing human prostacyclin synthase, which has been shown to attenuate MCT PAH.MethodsExpression levels of a panel of miRNAs were measured in MCT-PAH rats as compared to naïve (saline) control rats. Subsequently, MCT PAH rats were injected with a specific inhibitor (antagomiR) for miR-223 (A223) or a nonspecific control oligonucleotide (A-control) 4 days after MCT administration, then weekly. Three weeks later, RV systolic pressure and RV mass were measured. Total RNA, isolated from the lungs, microdissected pulmonary arteries, and right ventricle, was reverse transcribed and real-time quantitative PCR was performed. MiRNA levels were also measured in RNA isolated from paraffin sections of MCT-PAH rats overexpressing prostacyclin synthase.ResultsMiRs 17, 21, and 223 were consistently upregulated, whereas miRs 126, 145, 150, 204, 424, and 503 were downregulated in MCT PAH as compared to vehicle control. A223 significantly reduced levels of miR-223 in PA and lungs of MCT PAH rats as compared to levels measured in A-control or control MCT PAH rats, but A223 did not attenuate MCT PAH. Right ventricular mass and right ventricular systolic pressure in rats treated with A223 were not different from values in A-control or MCT PAH rats. In contrast, analysis of total RNA from lung specimens of MCT PAH rats overexpressing human prostacyclin synthase (hPGIS) demonstrated reversal of MCT-induced upregulation of miRs 17, 21, and 223 and an increase in levels of miR-424 and miR-503. Reduction in bone morphogenetic receptor 2 (BMPR2) messenger (m)RNA expression was not altered by A223, whereas human prostacyclin synthase overexpression restored BMPR2 mRNA to levels in MCT PAH to levels measured in naive controls.ConclusionsInhibition of miR-223 did not attenuate MCT PAH, whereas human prostacyclin synthase overexpression restored miRNA levels in MCT PAH to levels detected in naïve rats. These data may establish a paradigm linking attenuation of PAH to restoration of BMPR2 signaling.

CHRFAM7A, a human-specific and partially duplicated α7-nicotinic acetylcholine receptor gene with the potential to specify a human-specific inflammatory response to injury.

Conventional wisdom presumes that the α7nAChR product of CHRNA7 expression mediates the ability of the vagus nerve to regulate the inflammatory response to injury and infection. Yet, 15 years ago, a 2nd structurally distinct and human-specific α7nAChR gene was discovered that has largely escaped attention of the inflammation research community. The gene, originally called dupα7nAChR but now known as CHRFAM7A, has been studied exhaustively in psychiatric research because of its association with mental illness. However, dupα7nAChR/CHRFAM7A expression is relatively low in human brain but elevated in human leukocytes. Furthermore, α7nAChR research in human tissues has been confounded by cross-reacting antibodies and nonspecific oligonucleotide primers that crossreact in immunoblotting, immunohistochemistry, and RT-PCR. Yet, 3 independent reports show the human-specific CHRFAM7A changes cell responsiveness to the canonical α7nAChR/CHRNA7 ion-gated channel. Because of its potential for the injury research community, its possible significance to human leukocyte biology, and its relevance to human inflammation, we review the discovery and structure of the dupα7nAChR/CHRFAM7A gene, the distribution of its mRNA, and its biologic activities and then discuss its possible role(s) in specifying human inflammation and injury. In light of emerging concepts that point to a role for human-specific genes in complex human disease, the existence of a human-specific α7nAChR regulating inflammatory responses in injury underscores the need for caution in extrapolating findings in the α7nAChR literature to man. To this end, we discuss the translational implications of a uniquely human α7nAChR-like gene on new drug target discovery and therapeutics development for injury, infection, and inflammation.

DNA Stickers Promote Polymer Adsorption onto Cellulose

Adsorption of oligonucleotides onto model cellulose surfaces was investigated by comparing the Boese and Breaker's cellulose binding oligonucleotide (CBO) with a nonspecific oligonucleotide control (NSO). Measurements using the quartz crystal microbalance with dissipation technique confirmed that CBO adsorbed onto cellulose more than NSO, particularly at high ionic strengths (100 mM CaCl(2)). CBO showed a higher maximum adsorption on nanofibrillated and nanocrystalline cellulose than on regenerated cellulose, indicating a preference for the native cellulose I crystal structure under conditions that favored specific adsorption over calcium-mediated electrostatically driven adsorption. In addition, an anionic polyacrylamide (A-PAM) with grafted CBO also adsorbed onto the surface of cellulose in CaCl(2), whereas the unmodified A-PAM did not. This work shows that CBO performs as a "sticker", facilitating the adsorption of polyacrylamide onto cellulose, even under high ionic strength conditions where the adsorption of conventional polyelectrolytes is inhibited.

Mon1a Protein Acts in Trafficking through the Secretory Apparatus

Mon1a was originally identified as a modifier gene of vesicular traffic, as a mutant Mon1a allele resulted in increased localization of cell surface proteins, whereas reduced levels of Mon1a showed decreased secretory activity. Here we show that Mon1a affects different steps in the secretory pathway including endoplasmic reticulum-to-Golgi traffic. siRNA-dependent reduction of Mon1a levels resulted in a delay in the reformation of the Golgi apparatus after Brefeldin A treatment. Endoglycosidase H treatment of ts045VSVG-GFP confirmed that knockdown of Mon1a delayed endoplasmic reticulum-to-Golgi trafficking. Reductions in Mon1a also resulted in delayed trafficking from Golgi to the plasma membrane. Immunoprecipitation and mass spectrometry analysis showed that Mon1a associates with dynein intermediate chain. Reductions in Mon1a or dynein altered steady state Golgi morphology. Reductions in Mon1a delayed formation of ERGIC-53-positive vesicles, whereas reductions in dynein did not affect vesicle formation. These data provide strong evidence for a role for Mon1a in anterograde trafficking through the secretory apparatus.

Down-regulation of spinal d-amino acid oxidase expression blocks formalin-induced tonic pain

A series of inhibitors of d-amino acid oxidase (DAAO) are specific in blocking chronic pain, including formalin-induced tonic pain, neuropathic pain and bone cancer pain. This study used RNA interference technology to further validate the notion that spinal DAAO mediates formalin-induced pain. To target DAAO, a siRNA/DAAO formulated in polyetherimide (PEI) complexation and a shRNA/DAAO (shDAAO, with the same sequence as siRNA/DAAO after intracellular processing) expressed in recombinant adenoviral vectors were designed. The siRNA/DAAO was effective in blocking DAAO expression in NRK-52E rat kidney tubule epithelial cells, compared to the nonspecific oligonucleotides. Furthermore, multiple-daily intrathecal injections of both siRNA/DAAO and Ad-shDAAO for 7days significantly inhibited spinal DAAO expression by 50–80% as measured by real-time quantitative PCR and Western blot, and blocked spinal DAAO enzymatic activity by approximately 60%. Meanwhile, both siRNA/DAAO and Ad-shDAAO prevented formalin-induced tonic phase pain by approximately 60%. Multiple-daily intrathecal injections of siRNA/DAAO and Ad-shDAAO also blocked more than 30% spinal expression of GFAP, a biomarker for the activation of astrocytes. These results further suggest that down-regulation of spinal DAAO expression and enzymatic activity leads to analgesia with its mechanism potentially related to activation of astrocytes in the spinal cord.

Metal‐Induced Specific and Nonspecific Oligonucleotide Folding Studied by FRET and Related Biophysical and Bioanalytical Implications

Metal induced nucleic acid folding has been extensively studied with ribozymes, DNAzymes, tRNA and riboswitches. These RNA/DNA molecules usually have a high content of double-stranded regions to support a rigid scaffold. On the other hand, such rigid structural features are not available for many in vitro selected or rationally designed DNA aptamers; they adopt flexible random coil structures in the absence of target molecules. Upon target binding, these aptamers adaptively fold into a compact structure with a reduced end-to-end distance, making fluorescence resonance energy transfer (FRET) a popular signaling mechanism. However, nonspecific folding induced by mono- or divalent metal ions can also reduce the end-to-end distance and thus lead to false positive results. In this study we used a FRET pair labeled Hg(II) binding DNA and monitored metal-induced folding in the presence of various cations. While nonspecific electrostatically mediated folding can be very significant, at each tested salt condition, Hg(II) induced folding was still observed with a similar sensitivity. We also studied the biophysical meaning of the acceptor/donor fluorescence ratio that allowed us to explain the experimental observations. Potential solutions for this ionic strength problem have been discussed. For example, probes designed to signal the formation of double-stranded DNA showed a lower dependency on ionic strength.

Kinetics and Thermodynamics of Biotinylated Oligonucleotide Probe Binding to Particle-Immobilized Avidin and Implications for Multiplexing Applications

In this work, the kinetics and dissociation constant for the binding of a biotin-modified oligonucleotide to microparticle-immobilized avidin were measured. Avidin has been immobilized by both covalent coupling and bioaffinity capture to a surface prefunctionalized with biotin. The measured rate and equilibrium dissociation constants of avidin immobilized by these different methods have been compared with those for nonimmobilized avidin. We found that immobilization resulted in both a decrease in the rate of binding and an increase in the rate of dissociation leading to immobilized complexes having equilibrium dissociation constants of 7 ± 3 × 10(-12) M, higher than the value measured for the complex between biotin-modified oligonucleotide and nonimmobilized avidin and approximately 4 orders of magnitude larger than values for the wild-type avidin-biotin complex. Immobilized complex half-lives were found to be reduced to 5 days, which resulted in biotin ligands migrating between protein attached to different particles. Different immobilization methods showed little variation in complex stability but differed in total binding and nonspecific biotin-modified oligonucleotide binding. These findings are critical for the design of multiplexed assays where probe molecules are immobilized to biosensors via the avidin-biotin interaction.

Long Chain Acyl-CoA Synthetase-3 Is a Molecular Target for Peroxisome Proliferator-activated Receptor δ in HepG2 Hepatoma Cells

ACSL3 is a member of the long chain acyl-CoA synthetase (ACSL) family that plays key roles in fatty acid metabolism in various tissues in an isozyme-specific manner. Our previous studies showed that ACSL3 was transcriptionally up-regulated by the cytokine oncostatin M (OSM) in HepG2 cells, accompanied by reduced cellular triglyceride content and enhanced beta-oxidation. In this study, we investigated the molecular mechanism underlying the OSM-induced activation of ACSL3 gene transcription in HepG2 cells. We showed that OSM treatment resulted in a coordinated elevation of mRNA levels of ACSL3 and peroxisome proliferator-activated receptor delta (PPARdelta). The effect of OSM on ACSL3 mRNA expression was inhibited by cellular depletion of PPARdelta. By utilizing a PPARdelta agonist, L165041, we demonstrated that activation of PPARdelta led to increases in ACSL3 promoter activity, mRNA level, and protein level in HepG2 cells. Analysis of the ACSL3 promoter sequence identified two imperfect PPAR-responsive elements (PPRE) located in the ACSL3 promoter region -944 to -915, relative to the transcription start site. The up-regulation of ACSL3 promoter activity by PPARdelta was abolished by deletion of this PPRE-containing region or mutation to disrupt the binding sites. Direct interactions of PPARdelta with ACSL3-PPRE sequences were demonstrated by gel mobility shift and chromatin immunoprecipitation assays. Finally, we provided in vivo evidence showing that activation of PPARdelta by L165041 in hamsters increased ACSL3 mRNA and protein levels in the liver. These new findings define ACSL3 as a novel molecular target of PPARdelta in HepG2 cells and provide a regulatory mechanism for ACSL3 transcription in liver tissue.

Role of KLF4 and KLF12 in γ-Globin Gene Regulation.

Abstract 4075Poster Board III-1010Kruppel-like factors (KLFs) are a family of Cys2His2 zinc-finger DNA binding proteins that regulate gene expression through CACCC/GC/GT box binding in gene promoters. The CACCC element is critical for the developmental regulation of the human γ-globin and β-globin genes and studies are being done to ferret out various factors that bind this region and modulate gene activity. We recently identified two Kruppel-like factors, KLF4 and KLF12 whose expression levels decreased based on microarray-based gene profiling, concomitantly with decreased γ-globin expression during erythroid maturation. Decreased expression of both factors was further confirmed using quantitative PCR (qPCR) analysis. KLF4 and KLF12 mRNA levels decreased 56-fold and 16-fold respectively by day 28 compared to levels in day 7 erythroid progenitors. We next determined if KLF4 and KLF12 bind the γ-globin CACC box by electrophoretic mobility shift assay (EMSA) using nuclear proteins extracted from K562 cells and a [γ-32P] labeled γ-CACC probe located between -155 to -132 relative to the γ-globin gene cap site. Three DNA-protein complexes were observed. The specificity of these interactions was confirmed by competition reactions in which preincubation with excess unlabelled γ-CACC oligonucleotide effectively abolished the formation of all DNA/protein complexes; addition of nonspecific oligonucleotide had no effect on binding activity. Addition of polyclonal KLF4 or KLF12 antibodies to the EMSA reaction resulted in a marked decrease in intensity of all DNA-protein complexes suggesting both KLF4 and KLF12 are present. Additional studies were performed to determine the effect of the known fetal hemoglobin inducer hemin on KLF gene expression in K562 cells. Hemin stimulated γ-globin transcription while increasing KLF4 and KLF12 66-fold and 4-fold respectively (p<0.05). Hemin treatment in KU812 erythroleukemia cells which actively transcribe both γ- and β-globin, also produced a 10-fold increase (p<0.05) in KLF4; KLF12 levels were not changed. Our preliminary data suggest these KLFs might play a role in γ-globin regulation. siRNA mediated gene silencing studies are underway to determine if KLF4 and/or KLF12 play a direct role in γ-globin gene regulation. This mechanism could provide important molecular targets for fetal hemoglobin reactivation. This will be highly significant towards developing therapeutic strategies for hemoglobinopathies like sickle cell anemia and β-thalassemia. Disclosures:No relevant conflicts of interest to declare.

Immunological cross-reactivity of mycobacterial topoisomerase I and divergence from other bacteria

Mycobacterium smegmatis topoisomerase I exhibits several distinctive characteristics among all topoisomerases. The enzyme is devoid of Zn2+ fingers found typically in other bacterial type I topoisomerases and binds DNA in a site-specific manner. Using polyclonal antibodies, we demonstrate the high degree of relatedness of the enzyme across mycobacteria but not other bacteria. This absence of cross-reactivity from other bacteria indicates that mycobacterial topoisomerase I has diverged from Escherichia coli and other bacteria. We have investigated further the immunological properties of the enzyme by raising a panel of monoclonal antibodies that recognises different antigenically active regions of the enzyme and binds it with widely varied affinity. Inhibition of a C-terminal domain-specific antibody binding by enzyme-specific and non-specific oligonucleotides suggests the possibility of using these monoclonal antibodies to probe the structure, function and in vivo role of the enzyme.

Cyclic AMP Inducible Early Repressor Mediates the Termination of Corticotropin Releasing Hormone Transcription in Hypothalamic Neurons

Elevations of inducible cAMP early repressor (ICER), the repressor isoform of the cAMP-responsive element modulator (CREM), are associated with protein binding to the corticotrophin releasing hormone (CRH) promoter and termination of CRH transcriptional responses to stress. To determine whether endogenous ICER production represses CRH transcription, we examined the effect of CREM siRNA on forskolin-stimulated ICER formation and CRH transcription in the hypothalamic cell line, 4B, and in primary cultures of hypothalamic neurons. Cotransfection of 4B cells with CREM siRNA and a CRH promoter-driven luciferase reporter gene markedly reduced the induction of ICER by forskolin and potentiated the stimulatory effect of forskolin on CRH promoter activity, compared with cells cotransfected with a nonspecific oligonucleotide. The role of ICER on endogenous CRH expression was studied in primary cultures of hypothalamic neurons by examining the effect of CREM siRNA on forskolin-induced primary transcript (CRH hnRNA) using intronic real-time PCR. As observed during stress in vivo, forskolin-stimulated CRH hnRNA was transient, increasing up to 60 min and declining to near basal values by 3 h. Transfection of CREM siRNA reduced forskolin-induced ICER by about 45% 48-h later and partially reversed the declining phase of CRH hnRNA production at 3 h. The data provide evidence that endogenous ICER formation is required for termination of CRH transcription and support the hypothesis that ICER is part of an intracellular feedback mechanism limiting the activation of CRH transcription during stress.

Statistical methods of background correction for Illumina BeadArray data

Advances in technology have made different microarray platforms available. Among the many, Illumina BeadArrays are relatively new and have captured significant market share. With BeadArray technology, high data quality is generated from low sample input at reduced cost. However, the analysis methods for Illumina BeadArrays are far behind those for Affymetrix oligonucleotide arrays, and so need to be improved. In this article, we consider the problem of background correction for BeadArray data. One distinct feature of BeadArrays is that for each array, the noise is controlled by over 1000 bead types conjugated with non-specific oligonucleotide sequences. We extend the robust multi-array analysis (RMA) background correction model to incorporate the information from negative control beads, and consider three commonly used approaches for parameter estimation, namely, non-parametric, maximum likelihood estimation (MLE) and Bayesian estimation. The proposed approaches, as well as the existing background correction methods, are compared through simulation studies and a data example. We find that the maximum likelihood and Bayes methods seem to be the most promising. Supplementary data are available at Bioinformatics online.

Role of SP transcription factors in hormone-dependent modulation of genes in MCF-7 breast cancer cells: microarray and RNA interference studies

17beta-estradiol (E(2)) binds estrogen receptor alpha (ESR1) in MCF-7 cells and increases cell proliferation and survival through induction or repression of multiple genes. ESR1 interactions with DNA-bound specificity protein (SP) transcription factors is a nonclassical genomic estrogenic pathway and the role of SP transcription factors in mediating hormone-dependent activation or repression of genes in MCF-7 cells was investigated by microarrays and RNA interference. MCF-7 cells were transfected with a nonspecific oligonucleotide or a cocktail of small inhibitory RNAs (iSP), which knockdown SP1, SP3, and SP4 proteins, and treated with dimethylsulfoxide or 10 nM E(2) for 6 h. E(2) induced 62 and repressed 134 genes and the induction or repression was reversed in approximately 62% of the genes in cells transfected with iSP (ESR1/SP dependent), whereas hormonal activation or repression of the remaining genes was unaffected by iSP (SP independent). Analysis of the ESR1/SP-dependent and SP-independent genes showed minimal overlap with respect to the GO terms (functional processes) in genes induced or repressed, suggesting that the different genomic pathways may contribute independently to the hormone-induced phenotype in MCF-7 cells.

Improvement of SSO-mediated gene repair efficiency by nonspecific oligonucleotides

Targeted gene repair mediated by single-stranded DNA oligonucleotides (SSOs) is a promising method to correct the mutant gene precisely in prokaryotic and eukaryotic systems. We used a HeLa cell line, which was stably integrated with mutant enhanced green fluorescence protein gene (mEGFP) in the genome, to test the efficiency of SSO-mediated gene repair. We found that the mEGFP gene was successfully repaired by specific SSOs, but the efficiency was only ∼0.1%. Then we synthesized a series of nonspecific oligonucleotides, which were single-stranded DNA with different lengths and no significant similarity with the SSOs. We found the efficiency of SSO-mediated gene repair was increased by 6-fold in nonspecific oligonucleotides-treated cells. And this improvement in repair frequency correlated with the doses of the nonspecific oligonucleotides, instead of the lengths. Our evidence suggested that this increased repair efficiency was achieved by the transient alterations of the cellular proteome. We also found the obvious strand bias that antisense SSOs were much more effective than sense SSOs in the repair experiments with nonspecific oligonucleotides. These results provide a fresh clue into the mechanism of SSO-mediated targeted gene repair in mammalian cells.

Proteinase-activated Receptor-2 Induces Cyclooxygenase-2 Expression through β-Catenin and Cyclic AMP-response Element-binding Protein

Chronic inflammation of mucosae is associated with an increased cancer risk. Tumorigenesis in these tissues is associated with the activity of some proteinases, cyclooxygenase-2 (COX-2), and beta-catenin. Serine proteinases participate in both inflammation and tumorigenesis through the activation of proteinase-activated receptor-2 (PAR(2)), which up-regulates COX-2 by an unknown mechanism. We sought to determine whether beta-catenin participated in PAR(2)-induced COX-2 expression and through what cellular mechanism. In A549 epithelial cells, we showed that PAR(2) activation increased COX-2 expression through the beta-catenin/T cell factor transcription pathway. This effect was dependent upon ERK1/2 MAPK, which inhibited the beta-catenin-regulating protein, glycogen synthase kinase-3beta, and induced the activity of the cAMP-response element-binding protein (CREB). Knockdown of CREB by small interfering RNA revealed that PAR(2)-induced beta-catenin transcriptional activity and COX-2 expression were CREB-dependent. A co-immunoprecipitation assay revealed a physical interaction between CREB and beta-catenin. Thus, PAR(2) up-regulated COX-2 expression via an ERK1/2-mediated activation of the beta-catenin/Tcf-4 and CREB pathways. These findings reveal new cellular mechanisms by which serine proteinases may participate in tumor development and are particularly relevant to cancers associated with chronic mucosal inflammation, where serine proteinases are abundant and COX-2 overexpression is a common feature.