Large Changes In Expression Research Articles

Impact of Rhg1 copy number variation on a soybean cyst nematode resistance transcriptional network.

Soybean yield loss due to soybean cyst nematode (SCN) infestation has a negative impact on the U.S. economy. Most SCN-resistant soybeans carry a common resistance locus (Rhg1), conferred by copy number variation of a 31.2-kb segment at the Rhg1 locus. To identify the effects of Rhg1 copy number on the plant prior to SCN infection, we investigated genome-wide expression profiles in isogenic Fayette plants carrying different copy numbers at the Rhg1 locus (9-11 copies), that confer different levels of resistance to SCN. We found that even small differences in copy number lead to large changes in expression of downstream defense genes. The co-expression network constructed from differentially expressed genes (DEGs) outside the Rhg1 locus revealed complex effects of Rhg1 copy number on transcriptional regulation involving signal transduction and ethylene-mediated signaling pathways. Moreover, we report a variation in expression levels of phytoalexin biosynthesis-related genes that is correlated with copy number, and the activation of different NBS-LRR gene sets, indicating a broad effect of copy number on defense responses. Using qRT-PCR time series during SCN infection, we validated the SCN responses of DEGs detected in the copy number comparison and showed a stable upregulation of genes related to phytoalexin biosynthesis in resistant Fayette lines during the early stages of the incompatible interaction between soybeans and SCN, before syncytium formation. These results suggest additional genes that could enhance Rhg1-mediated SCN resistance.

High-Throughput Transcriptomics of Water Extracts Detects Reductions in Biological Activity with Water Treatment Processes.

The presence of numerous chemical contaminants from industrial, agricultural, and pharmaceutical sources in water supplies poses a potential risk to human and ecological health. Current chemical analyses suffer from limitations, including chemical coverage and high cost, and broad-coverage in vitro assays such as transcriptomics may further improve water quality monitoring by assessing a large range of possible effects. Here, we used high-throughput transcriptomics to assess the activity induced by field-derived water extracts in MCF7 breast carcinoma cells. Wastewater and surface water extracts induced the largest changes in expression among cell proliferation-related genes and neurological, estrogenic, and antibiotic pathways, whereas drinking and reclaimed water extracts that underwent advanced treatment showed substantially reduced bioactivity on both gene and pathway levels. Importantly, reclaimed water extracts induced fewer changes in gene expression than laboratory blanks, which reinforces previous conclusions based on targeted assays and improves confidence in bioassay-based monitoring of water quality.

Pregnancy-associated systemic gene expression compared to a pre-pregnancy baseline, among healthy women with term pregnancies.

Pregnancy is known to induce extensive biological changes in the healthy mother. Little is known, however, about what these changes are at the molecular level. We have examined systemic expression changes in protein-coding genes and long non-coding (lnc) RNAs during and after pregnancy, compared to before pregnancy, among healthy women with term pregnancies. Blood samples were collected from 14 healthy women enrolled in our prospective pregnancy cohort at 7 time-points (before, during and after pregnancy). Total RNA from frozen whole blood was used for RNA sequencing. Following raw read alignment and assembly, gene-level counts were obtained for protein-coding genes and long non-coding RNAs. At each time-point, cell type proportions were estimated using deconvolution. To examine associations between pregnancy status and gene expression over time, Generalized Estimating Equation (GEE) models were fitted, adjusting for age at conception, and with and without adjusting for changes in cell type proportions. Fold-changes in expression at each trimester were examined relative to the pre-pregnancy baseline. Numerous immune-related genes demonstrated pregnancy-associated expression, in a time-dependent manner. The genes that demonstrated the largest changes in expression included several that were neutrophil-related (over-expressed) and numerous immunoglobulin genes (under-expressed). Estimated cell proportions revealed a marked increase in neutrophils, and less so of activated CD4 memory T cells, during pregnancy, while most other cell type proportions decreased or remained unchanged. Adjusting for cell type proportions in our model revealed that although most of the expression changes were due to changes in cell type proportions in the bloodstream, transcriptional regulation was also involved, especially in down-regulating expression of type I interferon inducible genes. Compared to a pre-pregnancy baseline, there were extensive systemic changes in cell type proportions, gene expression and biological pathways associated with different stages of pregnancy and postpartum among healthy women. Some were due to changes in cell type proportions and some due to gene regulation. In addition to providing insight into term pregnancy among healthy women, these findings also provide a "normal" reference for abnormal pregnancies and for autoimmune diseases that improve or worsen during pregnancy, to assess deviations from normal.

IDHwt Glioblastomas Show Opposing Resistance Mechanisms Across Patients in Response to Standard Treatment

Abstract AIMS Glioblastoma (GBM) is the most common primary malignant brain tumour in adults. Despite aggressive treatment, a resistant tumour recurs in practically all patients. We therefore aimed to better understand the mechanisms driving this treatment resistance through investigating changes in gene expression across pairs of primary and recurrent GBM tumours. METHOD We generated or acquired bulk tumour RNA sequencing data for primary and first recurrent tumours from 107 patients who received standard treatment. Differential expression analysis between primary and recurrent samples found that the most dysregulated genes were involved in neurodevelopment and neurodifferentiation. We therefore used a publicly available ChIP-seq database to identify DNA binding factors for which binding sites are enriched in the promotors of genes with the largest expression changes from primary to recurrent. RESULTS Jumonji and AT-Rich Interacting Domain 2 (JARID2) was the most strongly enriched for binding to promotors of dysregulated genes. 65 patients showed an up-regulation and 42 showed a down-regulation of genes bound by this protein. The same set of JARID2 bound genes were found to be dysregulated in each direction, and correlated with the largest source of variation between samples in their response to treatment. Further enrichment analyses indicated that ‘Up’ responders may resist treatment through reduced proliferation and increased interaction with the tumour microenvironment, whereas ‘Down’ responders instead rely on a shift to mesenchymal cell states. CONCLUSION These results indicate that GBM tumours can be split into two subtypes that transcriptionally reprogramme in different directions through treatment and may benefit from different treatment approaches.

Multiple Criteria Optimization (MCO): A gene selection deterministic tool in RStudio.

Identifying genes with the largest expression changes (gene selection) to characterize a given condition is a popular first step to drive exploration into molecular mechanisms and is, therefore, paramount for therapeutic development. Reproducibility in the sciences makes it necessary to emphasize objectivity and systematic repeatability in biological and informatics analyses, including gene selection. With these two characteristics in mind, in previous works our research team has proposed using multiple criteria optimization (MCO) in gene selection to analyze microarray datasets. The result of this effort is the MCO algorithm, which selects genes with the largest expression changes without user manipulation of neither informatics nor statistical parameters. Furthermore, the user is not required to choose either a preference structure among multiple measures or a predetermined quantity of genes to be deemed significant a priori. This implies that using the same datasets and performance measures (PMs), the method will converge to the same set of selected differentially expressed genes (repeatability) despite who carries out the analysis (objectivity). The present work describes the development of an open-source tool in RStudio to enable both: (1) individual analysis of single datasets with two or three PMs and (2) meta-analysis with up to five microarray datasets, using one PM from each dataset. The capabilities afforded by the code include license-free portability and the possibility to carry out analyses via modest computer hardware, such as personal laptops. The code provides affordable, repeatable, and objective detection of differentially expressed genes from microarrays. It can be used to analyze other experiments with similar experimental comparative layouts, such as microRNA arrays and protein arrays, among others. As a demonstration of the capabilities of the code, the analysis of four publicly-available microarray datasets related to Parkinson´s Disease (PD) is presented here, treating each dataset individually or as a four-way meta-analysis. These MCO-supported analyses made it possible to identify MMP9 and TUBB2A as potential PD genetic biomarkers based on their persistent appearance across each of the case studies. A literature search confirmed the importance of these genes in PD and indeed as PD biomarkers, which evidences the code´s potential.

Efficient Lightweight Attention Network for Face Recognition

Although face recognition has achieved great success due to deep learning, many factors may affect the quality of faces in the wild, such as pose changes, age variations, and light changes, which can seriously affect the performance of face recognition. In this work, an effective approach called Efficient Lightweight Attention Networks (ELANet) is proposed to address the challenge brought by the impacts of poses and ages on face recognition performance. First, similar local patches are particularly important when the geometry and appearance of a face change drastically. To alleviate this challenge, spatial attention is used to capture important locally similar patches and channel attention is employed to focus on features with different levels of importance. Furthermore, Efficient Fusion Attention (EFA) module is designed to achieve better performance, which can alleviate the computational effort required by fusing spatial and channel attention. Second, multi-scale features learning is necessary because pose or large expression changes can cause similar recognition regions to appear at different scales. For this purpose, pyramid multi-scale module is presented, which constructs a series of features at different scales via pooling operations. Third, to unite low-level local detail information with high-level semantic information, the features of different layers are fused by Adaptively Spatial Feature Fusion (ASFF) instead of simply utilizing addition or concatenation. Compared to recent lightweight networks, the ELANet improved performance by 1.83% and 2.17% on the CPLFW and VGG2_FP datasets, respectively, and by 0.92% on the CALFW dataset. The ELANet addresses the challenge regarding the impacts of poses and ages on face recognition performance with few parameters and computational effort and is suitable for embedded and mobile devices.

Chromosomal neighbourhoods allow identification of organ specific changes in gene expression.

Although most genes share their chromosomal neighbourhood with other genes, distribution of genes has not been explored in the context of individual organ development; the common focus of developmental biology studies. Because developmental processes are often associated with initially subtle changes in gene expression, here we explored whether neighbouring genes are informative in the identification of differentially expressed genes. First, we quantified the chromosomal neighbourhood patterns of genes having related functional roles in the mammalian genome. Although the majority of protein coding genes have at least five neighbours within 1 Mb window around each gene, very few of these neighbours regulate development of the same organ. Analyses of transcriptomes of developing mouse molar teeth revealed that whereas expression of genes regulating tooth development changes, their neighbouring genes show no marked changes, irrespective of their level of expression. Finally, we test whether inclusion of gene neighbourhood in the analyses of differential expression could provide additional benefits. For the analyses, we developed an algorithm, called DELocal that identifies differentially expressed genes by comparing their expression changes to changes in adjacent genes in their chromosomal regions. Our results show that DELocal removes detection bias towards large changes in expression, thereby allowing identification of even subtle changes in development. Future studies, including the detection of differential expression, may benefit from, and further characterize the significance of gene-gene neighbour relationships.

Abstract MP35: Single-cell Analysis Uncovers Multiple Adventitial Fibroblast Populations With Expression Of CAD GWAS Genes In Murine Atherosclerosis

The arterial adventitia is a layer of cells outside the external lamina of blood vessels that contribute to the progression of coronary artery disease (CAD) like atherosclerosis. Adventitial fibroblasts are known regulators of vascular remodeling through their deposition of collagen fibrils around vessels during atherosclerotic lesion development. However, the molecular identity and function of the participating fibroblasts have not been well studied. We used single cell RNA sequencing (scRNA-seq) to characterize the transcriptome of adventitial fibroblasts and other cell types at multiple timepoints during atherogenesis in hyperlipidemic Ldlr knockout mice. Specifically, scRNA-seq was applied to Ldlr KO aortic adventitia after 0, 9, and 16 weeks of western diet (WD) feeding. Unbiased clustering analysis uncovered 9 different cell clusters, including 5 different fibroblast populations (Fibro1-5), and a single cluster each of macrophages, T cells, SMCs, and ECs. We observed that, as atherosclerosis progressed from 9 to 16 weeks of feedings, the proportion of total cells that were Fibro1, 3, and 4 decreased (33, 40, and 13%), while populations of Fibro2 and 5 slightly increased (13 and 18%). All of non-fibroblast populations (Macrophage, T cells, SMCs and ECs) increased from 9-16wks of diet. Next, we asked whether genes associated with coronary artery disease by human genome-wide association studies (GWAS) are expressed in different adventitial fibroblast clusters. We determined the expression of 266 genes implicated in CAD by GWAS and found 201 genes (76%) were expressed in adventitial cells at either/both 9 or 16wks. 34 genes displayed large changes in expression from 9 to 16wks. We identified five genes that were highly expressed in the adventitial Fibro5 cluster specifically, and additional other CAD GWAS genes specifically expressed in adventitial macrophages, SMCs, and ECs. In summary, we present here a cell atlas defined by scRNA-seq that reveals the heterogeneity amongst the fibroblasts in adventitial cells during atherogenesis. Additionally, we find that many CAD GWAS genes are expressed in adventitial cell populations, raising the intriguing possibility that CAD GWAS are identifying adventitial mechanisms that contribute to CAD.

Pressure-induced changes on the morphology and gene expression in mammalian cells.

ABSTRACTWe evaluated the effect of high hydrostatic pressure on mouse embryonic fibroblasts (MEFs) and mouse embryonic stem (ES) cells. Hydrostatic pressures of 15, 30, 60, and 90 MPa were applied for 10 min, and changes in gene expression were evaluated. Among genes related to mechanical stimuli, death-associated protein 3 was upregulated in MEF subjected to 90 MPa pressure; however, other genes known to be upregulated by mechanical stimuli did not change significantly. Genes related to cell differentiation did not show a large change in expression. On the other hand, genes related to pluripotency, such as Oct4 and Sox2, showed a twofold increase in expression upon application of 60 MPa hydrostatic pressure for 10 min. Although these changes did not persist after overnight culture, cells that were pressurized to 15 MPa showed an increase in pluripotency genes after overnight culture. When mouse ES cells were pressurized, they also showed an increase in the expression of pluripotency genes. These results show that hydrostatic pressure activates pluripotency genes in mammalian cells. This article has an associated First Person interview with the first author of the paper.

Identification and Expression Analyses of the Special 14-3-3 Gene Family in Papaya and its Involvement in Fruit Development, Ripening, and Abiotic Stress Responses.

Plant 14-3-3 proteins play key roles in regulating growth, development, and stress responses. However, little is known about this gene family in papaya (Carica papaya L.). We characterized eight 14-3-3 genes from the papaya genome and designed them as CpGRF1-8. Based on phylogenetic, conserved motif, and gene structure analyses, papaya CpGRFs were divided into ε and non-ε groups. Expression analysis showed differential and class-specific transcription patterns in different organs. Quantitative real-time polymerase chain reaction analysis showed that most CpGRFs had large changes in expression during fruit development and ripening. This indicated that the CpGRFs were involved in regulating fruit development and ripening. Significant expression changes occurred after cold, salt, and drought treatments in papaya seedlings, indicating that CpGRFs were also involved in signaling responses to abiotic stress. These results provide a transcription profile of 14-3-3 genes in organs, during fruit development and ripening and in response to stress. Some highly expressed, fruit-specific, and stress-responsive candidate CpGRFs will be identified for further genetic improvement of papayas.

Immune determinants of Barrett's progression to esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) develops from Barrett’s esophagus (BE), a chronic inflammatory state that can progress through a series of transformative dysplastic states before tumor development. While molecular and genetic changes of EAC tumors have been studied, immune microenvironment changes during Barrett’s progression to EAC remain poorly understood. In this study, we identify potential immunologic changes that can occur during BE-to-EAC progression. RNA sequencing (RNA-Seq) analysis on tissue samples from EAC patients undergoing surgical resection demonstrated that a subset of chemokines and cytokines, most notably IL6 and CXCL8, increased during BE progression to EAC. xCell deconvolution analysis investigating immune cell population changes demonstrated that the largest changes in expression during BE progression occurred in M2 macrophages, pro–B cells, and eosinophils. Multiplex immunohistochemical staining of tissue microarrays showed increased immune cell populations during Barrett’s progression to high-grade dysplasia. In contrast, EAC tumor sections were relatively immune poor, with a rise in PD-L1 expression and loss of CD8+ T cells. These data demonstrate that the EAC microenvironment is characterized by poor cytotoxic effector cell infiltration and increased immune inhibitory signaling. These findings suggest an immunosuppressive microenvironment, highlighting the need for further studies to explore immune modulatory therapy in EAC.

The genetic paradigms of dietary restriction fail to extend life span in cep-1(gk138) mutant of C. elegans p53 due to possible background mutations.

Dietary restriction (DR) increases life span and improves health in most model systems tested, including non-human primates. In C. elegans, as in other models, DR leads to reprogramming of metabolism, improvements in mitochondrial health, large changes in expression of cytoprotective genes and better proteostasis. Understandably, multiple global transcriptional regulators like transcription factors FOXO/DAF-16, FOXA/PHA-4, HSF1/HSF-1 and NRF2/SKN-1 are important for DR longevity. Considering the wide-ranging effects of p53 on organismal biology, we asked whether the C. elegans ortholog, CEP-1 is required for DR-mediated longevity assurance. We employed the widely-used TJ1 strain of cep-1(gk138). We show that cep-1(gk138) suppresses the life span extension of two genetic paradigms of DR, but two non-genetic modes of DR remain unaffected in this strain. We find that two aspects of DR, increased autophagy and up-regulation of the expression of cytoprotective xenobiotic detoxification program (cXDP) genes, are dampened in cep-1(gk138). Importantly, we find that background mutation(s) in the strain may be the actual cause for the phenotypic differences that we observed and cep-1 may not be directly involved in genetic DR-mediated longevity assurance in worms. Identifying these mutation(s) may reveal a novel regulator of longevity required specifically by genetic modes of DR.

Chronic ingestion of Primex-Z, compared with other common fat sources, drives worse liver injury and enhanced susceptibility to bacterial infections.

The aim of this study was to investigate putative different outcomes on the development of non-alcoholic fatty liver disease in mice using fat options regularly used in human nutrition. Male C57BL/6 mice were fed a control diet, and four different high-fat diets (HFD: 40% calories from fat; Research Diet, Inc., New Brunswick, New Jersey, USA) for 16 and 30 wk. HFDs had different common fat sources, including trans-fat, non-trans-fat palm oil (Primex-Z), palm oil alone, and corn oil alone. Mice were sacrificed and samples were collected for analysis. Using an unprecedented combination of in vivo imaging with immunometabolic phenotyping, we revealed that a HFD induced a major increase in hepatic lipid droplet deposition compared with control mice, being significantly higher in Primex-Z-fed mice. All HFD mice had similar or less weight gain as control mice; however, Primex-Z ingestion led to a higher increase in adiposity index (~90% increase) compared with other fat sources. Gene expression of isolated liver immune cells revealed large changes in expression of several inflammatory pathways, which were also more elevated in Primex-Z-fed mice, including Tnf (~20-fold), Il1b (~60-fold), and Tgfb (2.5-fold). Immunophenotyping and in vivo analysis showed that the frequency of hepatic immune cells was also disturbed during different HFD contents, rendering not only Kupffer cell depletion, but also reduced bacterial arresting ability. Different fat dietary sources imprint different immune and metabolic effects in the liver during consumption of an HFD. The present data highlighted that Primex-Z-a novel non-trans-fat-is not only able to damage hepatocytes, but also to impair liver ability to clear blood-borne infections.

Identification and Functional Validation of the miRNAs and their Targets, which are Essential to the Induction of Neointimal Hyperplasia

Angioplasty and stent procedures for widening the narrowed artery of atherosclerosis can cause restenosis by damaging endothelial cells. The main reason for this phenomenon is the migration and proliferation of vascular smooth muscle cells toward the intimal layer, which is called neointimal hyperplasia. After the function of miRNA, which is cell‐specific and fine‐tunes the translation of several mRNA targets, was known, several studies have identified miRNAs that affect arterial restenosis to overcome the limits of the previous drugs. However, most of the studies focused on one miRNA each. Therefore, it is necessary to ensure which of the individually studied miRNAs play a more important role at the beginning of restenosis. We should also ensure that there are any unknown miRNAs associated with restenosis regulation. In this study, we used the rat balloon injured carotid artery model to identify the entire differentially expressed (DE)‐miRNAs and DE‐mRNAs in the early stages of restenosis, through small RNA sequencing and total RNA sequencing, respectively. Among the DE‐miRNAs which show the same results in small RNA sequencing and RT‐qPCR, we focus on the rat, human conserved miRNAs with the largest change in expression (> 5 fold). The conserved DE‐miRNAs included previously well‐known miRNAs, such as miR‐221 and miR‐21, but also miRNAs, which show more change in expression but still unknown. By performing in vivo and in vitro functional assays, we were able to identify several undiscovered miRNAs that are effective in the cellular functions associated with the suppression of neointimal hyperplasia. Next, among the DE‐mRNAs identified by total RNA sequencing, we selected the predicted targets of the miRNAs through the target prediction databases (Targetscan, miRDB, miRmap). Also, we selected mRNAs functionally in context with the miRNAs by using the Gene Ontology (GO) term of the expected targets. We used RT‐qPCR to double‐check the changes in expression of these filtered targets and to confirm whether these predicted targets are actual targets for miRNAs. For the mRNA with the greatest variation, we performed luciferase assay to check whether the mRNA is a direct target of the miRNA. Additional functional assays were used to determine whether the target mRNA plays a central role in the progression of the neointimal hyperplasia.In summary, we identified entire miRNAs and their target mRNAs that are important for restenosis control. Through this study, we expect to utilize diverse miRNAs for the diagnosis and therapy of restenosis.Support or Funding InformationThis study was supported by the grant from the National Research Foundation of Korea (2018029571)

Reversible promoter methylation determines fluctuating expression of acute phase proteins.

Acute phase reactants (APRs) are secretory proteins exhibiting large expression changes in response to proinflammatory cytokines. Here we show that the expression pattern of a major human APR, that is C-reactive protein (CRP), is casually determined by DNMT3A and TET2-tuned promoter methylation status. CRP features a CpG-poor promoter with its CpG motifs located in binding sites of STAT3, C/EBP-β and NF-κB. These motifs are highly methylated at the resting state, but undergo STAT3- and NF-κB-dependent demethylation upon cytokine stimulation, leading to markedly enhanced recruitment of C/EBP-β that boosts CRP expression. Withdrawal of cytokines, by contrast, results in a rapid recovery of promoter methylation and termination of CRP induction. Further analysis suggests that reversible methylation also regulates the expression of highly inducible genes carrying CpG-poor promoters with APRs as representatives. Therefore, these CpG-poor promoters may evolve CpG-containing TF binding sites to harness dynamic methylation for prompt and reversible responses.

Underestimated Aspect of Mucopolysaccharidosis Pathogenesis: Global Changes in Cellular Processes Revealed by Transcriptomic Studies.

Mucopolysaccharidoses (MPS), a group of inherited metabolic disorders caused by deficiency in enzymes involved in degradation of glycosaminoglycans (GAGs), are examples (and models) of monogenic diseases. Accumulation of undegraded GAGs in lysosomes was supposed to be the major cause of MPS symptoms; however, their complexity and variability between particular types of the disease can be hardly explained by such a simple storage mechanism. Here we show that transcriptomic (RNA-seq) analysis of the material derived from fibroblasts of patients suffering from all types and subtypes of MPS, supported by RT-qPCR results, revealed surprisingly large changes in expression of genes involved in various cellular processes, indicating complex mechanisms of MPS. Although each MPS type and subtype was characterized by specific changes in gene expression profile, there were genes with significantly changed expression relative to wild-type cells that could be classified as common for various MPS types, suggesting similar disturbances in cellular processes. Therefore, both common features of all MPS types, and differences between them, might be potentially explained on the basis of changes in certain cellular processes arising from disturbed regulations of genes’ expression. These results may shed a new light on the mechanisms of genetic diseases, indicating how a single mutation can result in complex pathomechanism, due to perturbations in the network of cellular reactions. Moreover, they should be considered in studies on development of novel therapies, suggesting also why currently available treatment methods fail to correct all/most symptoms of MPS. We propose a hypothesis that disturbances in some cellular processes cannot be corrected by simple reduction of GAG levels; thus, combined therapies are necessary which may require improvement of these processes.

Differentially and Co-expressed Genes in Embryo, Germ-Line and Somatic Tissues of Tribolium castaneum.

Transcriptomic studies of Tribolium castaneum have led to significant advances in our understanding of co-regulation and differential expression of genes in development. However, previously used microarray approaches have covered only a subset of known genes. The aim of this study was to investigate gene expression patterns of beetle embryo, germ-line and somatic tissues. We identified 12,302 expressed genes and determined differentially expressed up and down-regulated genes among all samples. For example, 1624 and 3639 genes were differentially increased in expression greater than or equal to twofold change (FDR < 0.01) in testis vs. ovary (virgin female) and ovary vs. embryo (0-5 hr), respectively. Of these, many developmental, somatic and germ-line differentially expressed genes were identified. Furthermore, many maternally deposited transcripts were identified, whose expression either decreased rapidly or persisted during embryogenesis. Genes with the largest change in expression were predominantly decreased during early embryogenesis as compared to ovary or were increased in testis compared to embryo. We also identify zygotic genes induced after fertilization. The genome wide variation in transcript regulation in maternal and zygotic genes could provide additional information on how the anterior posterior axis formation is established in Tribolium embryos as compared to Drosophila. Together, our data will facilitate studies of comparative developmental biology as well as help identify candidate genes for identifying cis-elements to drive transgenic constructs.

MicroRNA-1246 regulates the radio-sensitizing effect of curcumin in bladder cancer cells via activating P53.

Radiotherapy is the primary option for bladder cancer patients, but it does not have obvious curative effects. This study was to investigate how to increase radiosensitivity in bladder cancer. The curcumin and irradiation treated T24 cells were used for analysis of microRNA expression (miRNA microarray), cell viability (Cell Proliferation Assay Kit), colony formation, apoptosis (Annexin V-FITC/7-AAD flow cytometry), miR-1246 and p53 mRNA (real-time PCR) and protein (Western blot) expression. Microarray assay identified 17 differentially expressed miRNAs (twofold change) in curcumin treated cells compared to control cells. Among them, miR-1246 was the miRNA with the largest change in expression after curcumin treatment. Curcumin significantly decreased T24 cell viability and colony formation in a concentration-dependent manner compared to control cells. miR-1246 expression was significantly higher in T24 cells than in SV-HUC-1 cells and the higher concentrations (10 or 20μM) of curcumin significantly down-regulated miR-1246 expression in T24 and HT-1376 cells. The combination of 10µM curcumin and irradiation was more effective in decreasing miR-1246 expression, cell viability and colony formation than curcumin or irradiation alone. Inhibition of miR-1246 significantly decreased cell viability and colony formation in T24 and HT-1376 cells. Transfection with antagomiR-1246 significantly increased the G0/G1-phase of T24 cells and induced apoptosis compared to cells transfected with antagomiR-NC. Luciferase reporter assay showed that the overexpression of miR-1246 suppressed the luciferase activity of the P53 3'-UTR reporter genes. miR-1246 is involved in the anti-cancer effects of curcumin and irradiation through targeting the inhibition of p53 gene translation in bladder cancer cells.

273-LB: Leukocyte-Endothelial Interaction Is Reduced by Insulin-Induced Downregulation of Endothelial CXCR4

An activated, pro-inflammatory endothelium is a key feature of obesity and type 2 diabetes and may contribute to adipose tissue inflammation, atherosclerosis and tumor formation. We have previously published a striking 4-fold increase in leukocyte adhesion to endothelium in mice with endothelial cell knockout of the insulin receptor. Here, we searched for insulin-regulated genes involved in leukocyte-endothelial cell interaction by performing RNA sequencing in primary human endothelial cells. Among genes with the largest change in expression after insulin treatment was C-X-C motif chemokine receptor 4 (CXCR4), which was downregulated by 54%. This reduction was completely prevented by pretreatment with wortmannin but not by U0126, an inhibitor of MAPK signaling. In CD31+ cells isolated by fluorescence-activated cell sorting of enzymatically dissociated tissue, CXCR4 mRNA in was 77% higher in mice with diet-induced obesity compared to lean controls and 37% higher in db/db mice compared to db/+ controls, consistent with upregulation of CXCR4 in endothelial cell insulin resistance. Stromal-derived factor-1 (SDF-1), the ligand for CXCR4, increased leukocyte adhesion to cultured endothelial cells. Insulin treatment of endothelial cells prevented 80% of this increase which in turn was abrogated by plerixafor, a clinically approved CXCR4 antagonist, or deletion of CXCR4 by gene editing. To study gain of endothelial cell insulin signaling in vivo, we examined transgenic mice with endothelial overexpression of insulin receptor substrate-1 (IRS-1). In vivo microscopy of mesenteric venules showed an increase in leukocyte rolling and adhesion after intravenous injection of SDF-1 in wildtype mice which was reduced by 83% in transgenic mice. We conclude that endothelial cell insulin signaling limits leukocyte-endothelial cell interaction through downregulation of CXCR4 and that targeting endothelial CXCR4 may protect against inflammation in insulin resistant states. Disclosure B. Kunkemoeller: None. T. Rathjen: Employee; Self; Bayer AG. J.C. Patti: None. Q. Li: None. K. Park: None. G.L. King: Research Support; Self; Sanofi. C. Rask-Madsen: None.

Effect of Aspirin on Placental Gene Expression in Preeclampsia

Preeclampsia (PreE) is a hypertensive disorder of pregnancy. It is the leading cause of maternal morbidity and mortality in pregnancy. Promisingly, aspirin provides some prevention for preeclampsia. The US Preventative Services Task Force (USPSTF) recommends low‐dose aspirin (81mg/day) as a preventative medication for women at high risk of PreE. However, the mechanism(s) by which aspirin prevents PreE are unknown.To better understand the mechanism by which aspirin prevents PreE, we compared gene expression of 84 cardiovascular genes involved in signal transduction, cardiac remodeling, ion transport, stress and immune response, apoptosis, sarcomere structure, transcriptional regulation, and cell cycle regulation (Qiagen RT2 Profiler Array) between placental tissues from 4 different cohorts: preeclamptic women, preeclamptic women who took aspirin during pregnancy, nonpreeclamptic women, and non‐preeclamptic women who took aspirin (N=3 per cohort). The placental tissues were obtained from the Maternal Fetal Tissue Bank at the University of Iowa (IRB# 200910784).We detected that 27 genes had a greater than 5 fold higher expression in the placentas of preeclamptics in comparison to placentas from women without PreE. However, 19 of these genes did not have elevated expression in either of the aspirin groups, even in women who developed PreE while taking aspirin. Further, only 1 gene (NPPB) met the 5 fold threshold when comparing women who developed PreE while on aspirin to women who developed PreE while not on aspirin. In comparing women who did not develop PreE while on aspirin to women without PreE who were not on aspirin, 2 genes were significantly elevated, but did not meet the 5 fold threshold (ACE and ADRA1A); yet, 2 genes were at least 5 fold higher in the group on aspirin (SLC12A1 and THBS1).To validate our approach, we interrogated a publicly available microarray dataset describing the expression changes in response to treatment with aspirin (GSE76583). Interestingly, others have also demonstrated that ACE, ADRA1A, SLC12A1, and THBS1 are up‐regulated in response to aspirin in primary colorectal carcinoma cells when treated with aspirin ex vivo. Although we have taken a different approach by studying samples from participants who took aspirin for clinical reasons, we identify some of the same targets. Thus, our approach to use patient samples may yield insights into the pathways by which aspirin prevents PreE. At this sample size, significant variability between participants makes the identification of statistically significant challenges difficult, but importantly we were still able to identify numerous genes that had a large change in expression with the use of aspirin. The identification of gene expression changes will be utilized to understand the mechanisms by which aspirin prevents PreE and to design preventative and therapeutic treatments to efficiently target these mechanisms.Support or Funding InformationThe American Physiological Society STRIDE fellowship (BC), The Iowa Center for Research by Undergraduates Fellowship (BC), AHA Postdoctoral Fellowship (SS), AHA Strategically Focused Research Network in Hypertension (MS, JG, SS, DS), and the Swift Family Foundation (DS, ED).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.