Numerous Gene Targets Research Articles

Chemical strategies for strand selection in short-interfering RNAs.

Therapeutic small interfering RNAs (siRNAs) are double stranded RNAs capable of potent and specific gene silencing through activation of the RNA interference (RNAi) pathway. The potential of siRNA drugs has recently been highlighted by the approval of multiple siRNA therapeutics. These successes relied heavily on chemically modified nucleic acids and their impact on stability, delivery, potency, and off-target effects. Despite remarkable progress, clinical trials still face failure due to off-target effects such as off-target gene dysregulation. Each siRNA strand can downregulate numerous gene targets while also contributing towards saturation of the RNAi machinery, leading to the upregulation of miRNA-repressed genes. Eliminating sense strand uptake effectively reduces off-target gene silencing and helps limit the disruption to endogenous regulatory mechanisms. Therefore, our understanding of strand selection has a direct impact on the success of future siRNA therapeutics. In this review, the approaches used to improve strand uptake are discussed and effective methods are summarized.

Impacts of multi-year field exposure of agricultural soil to macrolide antibiotics on the abundance of antibiotic resistance genes and selected mobile genetic elements

Exposure of environmental bacteria to antibiotics may be increasing the global resistome. Antibiotic residues are entrained into agricultural soil through the application of animal and human wastes, and irrigation with reclaimed water. The impact of a mixture of three macrolide antibiotics on the abundance of selected genes associated with antibiotic resistance and genetic mobility were determined in a long-term field experiment undertaken in London, Canada. Replicated plots received annual applications of a mixture of erythromycin, clarithromycin and azithromycin every spring since 2010. Each antibiotic was added directly to the soil at a concentration of either 0.1 or 10 mg kg soil−1 and all plots were cropped to soybeans. By means of qPCR, no gene targets were enriched in soil exposed to the 0.1 mg kg soil−1 dose compared to untreated control. In contrast, the relative abundance of several gene targets including int1, sul2 and mphE increased significantly with the annual exposure to the 10 mg kg soil−1 dose. By means of high-throughput qPCR, numerous gene targets associated with resistance to aminoglycosides, sulfonamides, trimethoprim, streptomycin, quaternary ammonium chemicals as well as mobile genetic elements (tnpA, IS26 and IS6100) were detected in soil exposed to 10 mg kg soil−1, but not the lower dose. Overall, exposure of soil to macrolide antibiotics increased the relative abundance of numerous gene targets associated with resistance to macrolides and other antibiotics, and mobile genetic elements. This occurred at an exposure dose that is unrealistically high, but did not occur at the lower more realistic exposure dose.

Inhibition of microRNA-124-3p as a novel therapeutic strategy for the treatment of Gulf War Illness: Evaluation in a rat model

Gulf War Illness (GWI) is a chronic, multisymptom illness that continues to affect up to 30% of veterans deployed to the Persian Gulf during the 1990–1991 Gulf War. After nearly 30 years, useful treatments for GWI are lacking and underlying cellular and molecular mechanisms involved in its pathobiology remain poorly understood, although exposures to pyridostigmine bromide (PB) and pesticides are consistently identified to be among the strongest risk factors. Alleviation of the broad range of symptoms manifested in GWI, which involve the central nervous system, the neuroendocrine system, and the immune system likely requires therapies that are able to activate and inactivate a large set of orchestrated genes. Previous work in our laboratory using an established rat model of GWI identified persistent elevation of microRNA-124-3p (miR-124) levels in the hippocampus whose numerous gene targets are involved in cognition-associated pathways and neuroendocrine function. This study aimed to investigate the broad effects of miR-124 inhibition in the brain 9 months after completion of a 28-day exposure regimen of PB, DEET (N,N-diethyl-3-methylbenzamide), permethrin, and mild stress by profiling the hippocampal expression of genes known to play a critical role in synaptic plasticity, glucocorticoid signaling, and neurogenesis. We determined that intracerebroventricular infusion of a miR-124 antisense oligonucleotide (miR-124 inhibitor; 0.05-0.5 nmol/day/28 days), but not a negative control oligonucleotide, into the lateral ventricle of the brain caused increased protein expression of multiple validated miR-124 targets and increased expression of downstream target genes important for cognition and neuroendocrine signaling in the hippocampus. Off-target cardiotoxic effects were revealed in GWI rats receiving 0.1 nmol/day as indicated by the detection in plasma of 5 highly elevated protein cardiac injury markers and 6 upregulated cardiac-enriched miRNAs in plasma exosomes determined by next-generation sequencing. Results from this study suggest that in vivo inhibition of miR-124 function in the hippocampus is a promising, novel therapeutic approach to improve cognition and neuroendocrine dysfunction in GWI. Additional preclinical studies in animal models to assess feasibility and safety by developing a practical, noninvasive drug delivery system to the brain and exploring potential adverse toxicologic effects of miR-124 inhibition are warranted.

Optimization of a microfluidics-based next generation sequencing assay for clinical oncology diagnostics.

Massively parallel, or next-generation, sequencing is a powerful technique for the assessment of somatic genomic alterations in cancer samples. Numerous gene targets can be interrogated simultaneously with a high degree of sensitivity. The clinical standard of care for many advanced solid and hematologic malignancies currently requires mutation analysis of several genes in the front-line setting, making focused next generation sequencing (NGS) assays an effective tool for clinical molecular diagnostic laboratories. We have utilized an integrated microfluidics circuit (IFC) technology for multiplex PCR-based library preparation coupled with a bioinformatic method designed to enhance indel detection. A parallel low input PCR-based library preparation method was developed for challenging specimens with low DNA yield. Computational data filters were written to optimize analytic sensitivity and specificity for clinically relevant variants. Minimum sequencing coverage and precision of variant calls were the two primary criteria used to establish minimum DNA mass input onto the IFC. Wet-bench and bioinformatics protocols were modified based on data from the optimization and familiarization process to improve assay performance. The NGS platform was then clinically validated for single nucleotide and indel (up to 93 base pair) variant detection with overall analytic accuracy of 98% (97% sensitivity; 100% specificity) using as little as 3 ng of formalin-fixed, paraffin-embedded DNA or 0.3 ng of unfixed DNA. We created a targeted clinical NGS assay for common solid and hematologic cancers with high sensitivity, high specificity, and the flexibility to test very limited tissue samples often encountered in routine clinical practice.

Dehydroleucodine Induces a TP73-dependent Transcriptional Regulation of Multiple Cell Death Target Genes in Human Glioblastoma Cells.

Dehydroleucodine, a natural sesquiterpene lactone from Artemisia douglassiana Besser (Argentine) and Gynoxys verrucosa (Ecuador). To define the molecular mechanisms underlying the effect of dehydroleucodine on the human glioblastoma cells. Various techniques (cDNA expression array, real-time quantitative PCR, chromatin immunprecipitation, luciferase reporter assay, use of phosphospecific antibodies, immunoprecipitation, immunoblotting, apoptosis and autophagy assays) were employed to define and validate multiple molecular gene targets affected in human glioblastoma cells upon dehydroleucodine exposure. Dehydroleucodine exposure upregulated the total and phosphorylated (p-Y99) levels of TP73 in U87- MG glioblastoma cells. We found that TP73 silencing led to a partial rescue of U87-MG cells from the cell death induced by dehydroleucodine. Upon the dehydroleucodine exposure numerous gene targets were upregulated and downregulated through a TP73-dependent transcriptional mechanism. Some of these gene targets are known to be involved in cell cycle arrest, apoptosis, autophagy and necroptosis. Dehydroleucodine induced the TP73 binding to the specific genes promoters (CDKN1A, BAX, TP53AIP1, CYLD, RIPK1, and APG5L). Moreover, the exposure of U87-MG cells to dehydroleucodine upregulated the protein levels of CDKN1A, BAX, TP53AIP1, CYLD, RIPK1, APG5L, and downregulated the CASP8 level. The formation of RIPK1 protein complexes and phosphorylation of MLKL were induced by dehydroleucodine supporting the notion of multiple cell death mechanisms implicated in the tumor cell response to dehydroleucodine. This multifaceted study led to a conclusion that dehydroleucodine induces the phosphorylation of tumor protein TP73 and in turn activates numerous TP73-target genes regulating apoptosis, autophagy and necroptosis in human glioblastoma cells..

225 VALIDATION OF REFERENCE microRNAs FOR NORMALIZING EXPRESSION DATA GENERATED BY QUANTITATIVE PCR

Small noncoding RNAs such as microRNAs (miRNA) act as posttranscriptional regulators of numerous gene targets. The level of expression of these epigenetic factors can regulate the stability and translation of target mRNA molecules. Quantification of miRNA expression levels can therefore help us understand biological processes such as those regulating oocyte and pre-implantation embryo development. To date, most studies that have examined miRNA expression levels have used expression of either reference mRNAs or U6 spliceosomal RNA or miRNA Let-7a for normalization. We analysed the suitability of several miRNAs as potential expression normalizers in bovine oocytes and early embryos. Bovine oocytes at the germinal vesicle and metaphase II stages, and zygotes, 2-cell, 4-cell and 8-cell embryos, morulae, and blastocysts were collected during three in vitro fertilization replicates. qPCR was performed to quantify expression of miR-93, miR-103a, miR-26a, miR-191, miR-23b, Let-7a, and U6. The average starting material for each sample was determined with the help of specific standard curves made for each primer set. Subsequently, geNorm software was used to identify a set of stably transcribed miRNAs. Surprisingly, U6 spliceosomal RNA and Let-7a, which are widely used to normalise miRNA expression levels, were not stably expressed and were therefore considered unsuitable for normalization. Stepwise removal to determine the optimal number of reference miRNAs identified miR-93 and miR-103a as the most stably expressed. It is concluded that the combination of miR-93 and miR-103a can be used for normalizing miRNA expression for qPCR experiments on bovine oocytes and pre-implantation embryos.

Nuclear glycolytic enzyme enolase of Toxoplasma gondii functions as a transcriptional regulator.

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii.

Clinical and biochemical function of polymorphic NR0B1 GGAA-microsatellites in Ewing sarcoma: a report from the Children's Oncology Group.

BackgroundThe genetics involved in Ewing sarcoma susceptibility and prognosis are poorly understood. EWS/FLI and related EWS/ETS chimeras upregulate numerous gene targets via promoter-based GGAA-microsatellite response elements. These microsatellites are highly polymorphic in humans, and preliminary evidence suggests EWS/FLI-mediated gene expression is highly dependent on the number of GGAA motifs within the microsatellite.ObjectivesHere we sought to examine the polymorphic spectrum of a GGAA-microsatellite within the NR0B1 promoter (a critical EWS/FLI target) in primary Ewing sarcoma tumors, and characterize how this polymorphism influences gene expression and clinical outcomes.ResultsA complex, bimodal pattern of EWS/FLI-mediated gene expression was observed across a wide range of GGAA motifs, with maximal expression observed in constructs containing 20–26 GGAA motifs. Relative to white European and African controls, the NR0B1 GGAA-microsatellite in tumor cells demonstrated a strong bias for haplotypes containing 21–25 GGAA motifs suggesting a relationship between microsatellite function and disease susceptibility. This selection bias was not a product of microsatellite instability in tumor samples, nor was there a correlation between NR0B1 GGAA-microsatellite polymorphisms and survival outcomes.ConclusionsThese data suggest that GGAA-microsatellite polymorphisms observed in human populations modulate EWS/FLI-mediated gene expression and may influence disease susceptibility in Ewing sarcoma.

A novel PCR-based approach to discover miRNA target genes.

MiRNAs are potent regulators of gene expression, and most miRNAs have from several to several thousands of gene targets. Validating the numerous gene targets of a given miRNA remains challenging despite the existence of various tools and databases that predict candidate gene-miRNA pairs. In the present study, we present a high-throughput but flexible method that applies a PCR-based application to simulate the binding of miRNAs to their gene targets. Using hsa-miR-377 as an illustrative example, our method was able to identify 13 potential targets of hsa-miR-377. Moreover, our results include 2 genes (SOD2 and PPM1A) that have already been verified as targets of hsa-miR-377. Our method may provide an alternative way of identifying the gene targets of miRNAs for future research.

Microarray analysis revealed dysregulation of multiple genes associated with chemoresistance to As 2O 3 and increased tumor aggressiveness in a newly established arsenic-resistant ovarian cancer cell line, OVCAR-3/AsR

The potential of arsenic trioxide (As 2O 3) for use as a novel therapy for ovarian cancer treatment has been increasingly recognized. In this study, we developed an arsenic-resistant OVCAR-3 subline (OVCAR-3/AsR) and aimed to identify the molecular mechanisms and signaling pathways contributing to the development of acquired arsenic chemoresistance in ovarian cancer. OVCAR-3/AsR cells were obtained following continual exposure of parental OVCAR-3 cells to low dose As 2O 3 for 12 months. Cytotoxicity of OVCAR-3/AsR cells to As 2O 3, paclitaxel and cisplatin was investigated. Cell apoptosis and cell cycle distribution following As 2O 3 treatment of OVCAR-3/AsR cells was also analyzed using flow cytometry. Subsequently, cDNA microarray analysis was performed from the RNA samples of OVCAR-3 and OVCAR-3/AsR cells in duplicate experiments. Microarray data were analyzed using Genespring® and Pathway Studio® Softwares. OVCAR-3/AsR cells showed 9-fold greater resistance to As 2O 3 and lack of collateral resistance to cisplatin and paclitaxel. Compared with parental OVCAR-3 cells, OVCAR-3/AsR had significantly lower apoptotic rates following As 2O 3 treatment. These cells were also arrested at both the S phase and G 2/M phase of the cell cycle after exposure to high concentrations of As 2O 3. Gene expression profiling revealed significant differences in expression levels of 397 genes between OVCAR-3/AsR and OVCAR-3 cells. The differentially regulated transcripts genes have functional ontologies related to continued cancer cell growth, cell survival, tumor metastasis and tumor aggressiveness. Additionally, numerous gene targets of the nuclear factor erythroid 2-related factor 2 (NRF2) transcription factor showed elevated expression in OVCAR-3/AsR cells. Subsequent pathway analysis further revealed a gene network involving interleukin 1-alpha (IL1A) in mediating the arsenic-resistant phenotype. These results showed that changes in multiple genes and an increased in tumor aggressiveness occurred during the development of acquired chemoresistance to As 2O 3 in ovarian cancer cells. The functional relevance of these genetic changes should be validated in future studies.