Fold Change In Expression Research Articles

Toll-Like Receptor Agonists Modulate Galectin Gene Expression in Goat Blood

Pathogenesis and lack of effective therapeutics pose a threat to animal and public health. Recognition of pathogen-associated molecular patterns (PAMPS) by Toll-like receptors is associated with gene activation. Galectins are sugar-binding proteins that play a role in pathogen-recognition and immune modulation. The objective of this study was to investigate the effect of different TLR agonists on galectin gene expression and secretion in goat blood. Blood was collected from the jugular vein of BoerXSpanish goats (n=3). Blood was treated with 10 µg/ml Lipopolysaccharide, Peptidoglycan, 12.5 µg/ml of Polyinosinic-polycytidylic acid, 10 µg/ml CpG ODN (2216) and 10 µg/ml CpG ODN (2006) or PBS control. Secretion of galectin-1, -3 and -9 was measured by ELISA. Expression of LGALS-1, -2, -3, -4, -7, -8, -9, -11, -12, -14, -15, -16 was assessed using real-time PCR. Beta-actin and GAPDH served as controls. Fold change in gene expression was calculated using the 2−ΔΔCt method. Data were analyzed using SAS 9.4 statistical analysis software (p < 0.05). Expression and secretion galectins was modulated by different PAMPS (p < 0.05) and may have implications for design of therapeutics based on TLR agonists.

Alterations in Gastric Mucosal Expression of Calcitonin Gene-Related Peptides, Vanilloid Receptors, and Heme Oxygenase-1 Mediate Gastroprotective Action of Carbon Monoxide against Ethanol-Induced Gastric Mucosal Lesions.

Carbon monoxide (CO) has been reported to contribute to the maintenance of gastric mucosal integrity, gastroprotection, and ulcer healing. However, involvement of transient receptor potential vanilloid receptor type 1 (TRPV1) located on afferent sensory fibers endings and sensory neuropeptide calcitonin gene-related peptide (CGRP) in CO-mediated gastroprotection against ethanol-induced gastric damage has not been explored. Male Wistar rats with and without denervation of afferent sensory neurons induced by capsaicin (total dose 125 mg/kg within 3 days) were pretreated with vehicle, CO donor tricarbonyldichlororuthenium (II) dimer (CORM-2, 5 mg/kg i.g.), administered alone or with CGRP-α (10 μg/kg i.p.) or TRPV1 antagonist capsazepine (5 mg/kg i.g.), followed 30 min later by intragastric (i.g.) administration of 75% ethanol. The area of gastric damage and gastric blood flow (GBF) were assessed planimetrically and by laser flowmetry, respectively. Microscopic evaluation of ethanol-induced gastric lesions was performed after haematoxylin/eosin (H&E) or alcian blue/periodic acid-Schiff/alcian blue (AB/PAS) staining. Gastric mucosal mRNA fold change for heme oxygenase (HMOX)-1, HMOX-2, CGRP-α, CGRP-β, inducible nitric oxide synthase (iNOS), endothelial (e)NOS, neuronal (n)NOS, cyclooxygenase (COX)-1, COX-2, and protein expression for HMOX-1 and TRPV1 was determined by real-time PCR or Western blot, respectively. Pretreatment with CORM-2 combined or not with CGRP reduced ethanol-induced gastric lesions and elevated GBF. Capsaicin-denervation or co-treatment with capsazepine or CGRP and CORM-2 in capsaicin-denervated animals failed to affect these beneficial effects of CO donor. In rats with intact sensory nerves, CORM-2 increased gastric mRNA level for HMOX-1 and CGRP-α. In capsaicin-denervated rats, CORM-2 increased eNOS mRNA fold change and TRPV1 protein expression while capsaicin denervation itself decreased HMOX-1 protein expression and eNOS mRNA level. We conclude that CO prevents gastric mucosa from ethanol-induced lesions due to activation of TRPV1/CGRP-α system and accompanying increase in gastric microcirculation but independently on afferent sensory nerve activity despite the stimulation of TRPV1 protein and CGRP-α mRNA expression.

Expression of deiodinase 2 (DIO2) and integrin alpha 9 (ITGA9) genes as indicators' of adaptability and their relationship with physio-biochemical parameters in Tharparkar and Karan Fries heifers during different seasons

The aim of the study was to observe the influence of ambient conditions (seasons) on expression pattern of deiodinase 2 (DIO2) and integrin alpha 9 (ITGA9) genes in peripheral blood mononuclear cells (PBMC) and their relationship with physiological and biochemical parameters of Tharparkar and Karan Fries heifers. Healthy heifers of Tharparkar and Karan Fries breed (6 each) were selected and blood samples were collected from these animals at weekly interval. These blood samples were centrifuged to separate the plasma (for biochemical parameters) and buffy coat (for total RNA isolation). The expression of DIO2 gene was down-regulated during hot humid season and up-regulated during winter season in both the breeds. The DIO2 gene showed a positive correlation with thyroid hormones (T3, T4) and negative correlation with physiological responses (RR, PR, RT, ST), cortisol hormone, antioxidant enzymes (SOD, GPx) and non-esterified fatty-acids (NEFA). The expression pattern of ITGA9 gene was opposite to DIO2 gene, i.e. up-regulated during hot humid season and down-regulated during winter season in both the breeds. ITGA9 gene expression showed a positive correlation with cortisol, antioxidant enzymes, NEFA and physiological responses, and negative correlation with thyroid hormones. The fold change in expression of DIO2 and ITGA9 genes was higher in Karan Fries than Tharparkar heifers during hot humid and winter season. The dry matter intake was lower during hot-humid season than autumn and winter season in both the breeds of heifer. The expression pattern of the DIO2 and ITGA9 genes and their relationship with physio-biochemical parameters revealed the essential role in adaptability of cattle as candidate gene. Based on the results of present study, it can be stated that Tharparkar heifers are better adapted to tropical climatic conditions than Karan Fries heifers.

The role of miRNAs 34a, 146a, 320a and 542 in the synergistic anticancer effects of methyl 2-(5-fluoro-2-hydroxyphenyl)-1H- benzo[d]imidazole-5-carboxylate (MBIC) with doxorubicin in breast cancer cells.

Combination Index (CI) analysis suggested that MBIC and doxorubicin synergistically inhibited up to 97% of cell proliferation in ER+/PR+MCF-7 and triple negative MDA-MB-231 breast cancer cell lines. Moreover, treatment of the breast cancer cells with the combined drugs resulted in lower IC50 values in contrast to the individual drug treatment. Small noncoding microRNAs (miRNA) may function as non-mutational gene regulators at post-transcriptional level of protein synthesis. In the present study, the effect of the combined treatment of MBIC and doxorubicin on the expression level of several miRNAs including miR-34a, miR-146a, miR-320a and miR-542 were evaluated in MCF-7 and MDA-MB-231 breast cancer cell lines. These miRNAs have the potential to alter the protein level of survivin, the anti-apoptotic protein and reduce the metastatic activity in human breast cancer cell lines by interfering with the nuclear accumulation of NF-κB. Our results demonstrated the several fold changes in expression of miRNAs, which is drug and cell line dependent. This finding demonstrated a functional synergistic network between miR-34a, miR-320a and miR-542 that are negatively involved in post-transcriptional regulation of survivin in MCF-7 cells. While in MDA-MB-231 cells, changes in expression level of miR-146a was correlated with inhibition of the nuclear translocation of NF-κB. The overall result suggested that alteration in protein level and location of survivin and NF-κB by miR-34a, miR-320a, miR-146a and miR-542, remarkably influenced the synergistic enhancement of combined MBIC and doxorubicin in treatment of aggressive and less aggressive human breast cancer cell lines.

TNFRSF10C methylation is a new epigenetic biomarker for colorectal cancer.

BackgroundAbnormal methylation of TNFRSF10C was found to be associated with different types of cancers, excluding colorectal cancer (CRC). In this paper, the performance of TNFRSF10C methylation in CRC was studied in two stages.MethodThe discovery stage was involved with 38 pairs of CRC tumor and paired adjacent non-tumor tissues, and 69 pairs of CRC tumor and paired adjacent non-tumor tissues were used for the validation stage. Quantitative methylation specific PCR (qMSP) method and percentage of methylated reference (PMR) were used to test and represent the methylation level of TNFRSF10C, respectively. A dual-luciferase reporter gene experiment was conducted to evaluate the promoter activity of TNFRSF10C fragment.ResultsA significant association of TNFRSF10C promoter hypermethylation with CRC was found and validated (discovery stage: 24.67 ± 7.52 vs. 3.36 ± 0.89; P = 0.003; validation stage: 31.21 ± 12.48 vs. 4.52 ± 1.47; P = 0.0005). Subsequent analyses of TCGA data among 46 pairs of CRC samples further confirmed our findings (cg23965061: P = 4E − 6; cg14015044: P = 1E − 7). Dual-luciferase reporter gene assay revealed that TNFRSF10C fragment was able to significantly promote gene expression (Fold change = 2.375, P = 0.013). Our data confirmed that TNFRSF10C promoter hypermethylation can predict shorter overall survival of CRC patients (P = 0.032). Additionally, bioinformatics analyses indicated that TNFRSF10C hypermethylation was significantly associated with lower TNFRSF10C expression.ConclusionOur work suggested that TNFRSF10C hypermethylation was significantly associated with the risk of CRC.

Evaluation and validation of housekeeping genes in two contrast species of thyme plant to drought stress using real-time PCR

To decrease errors and increase accuracy and reliability of quantitative real-time PCR (qRT-PCR) results, the use of a reference gene is inevitable. Despite the industrial importance of genus Thymus, not any validated reference gene has not been reported for T. kotschyanus and T. vulgaris which could limit such investigations. In this study, the expression stability of seven housekeeping genes including Actin, Cyclophilin-18, elongation factor-1A, glyceraldehyde-3-phosphate dehydrogenase, 18S ribosomal RNA, Cullin, and Polypyrimidine tract-binding protein were evaluated in T. kotschyanus and T. vulgaris which grown at four levels of drought stress using geNorm, NormFinder, and BestKeeper algorithms. Histone deacetylase-6 (HDA-6) gene was also used for validation of evaluated reference genes. In T. vulgaris, all of the algorithms similarly ranked elongation factor-1A and glyceraldehyde-3-phosphate dehydrogenase as the two most stably expressed genes. In T. kotschyanus, only NormFinder and BestKeeper had a similar ranking and identified Actin and glyceraldehyde-3-phosphate dehydrogenase as the two most stably expressed genes, but geNorm algorithm ranked elongation factor-1A and glyceraldehyde-3-phosphate dehydrogenase as the best two reference genes. On the other hand, all algorithms ranked 18S rRNA and Cyclophilin-18 as the least stable genes in T. kotschyanus and T. vulgaris, respectively. Validation results indicated that there was a significant change (0.53–3.19 fold change) in relative expression of HDA-6 normalized by the best stable gene compare to the least ranked gene. Our study presented the first systematic validation of reference gene(s) selection in T. kotschyanus and T. vulgaris and provided useful information to obtain more accurate qRT-PCR results in these species.

Expression pattern of new genes regulating female sex differentiation and in vitro maturational status of oocytes in pigs

The processes underlying maturation of mammalian oocytes are considered crucial for the oocytes ability to undergo monospermic fertilization. The same factors of influence are suggested to impact the development of sex associated characteristics, allowing sex differentiation to progress during embryonic growth. The primary aim of the study was to analyze the gene ontology groups involved in regulation of porcine oocytes' response to endogenous stimuli. The results obtained would indicate potential genes influencing sex differentiation. Additionally, they could help to determine new genetic markers, expression profile of which is substantially regulated during porcine oocytes’ in vitro maturation. To achieve that, porcine oocytes were collected for analysis before and after in vitro maturation. Pigs were used as they are a readily available model that presents significant similarity to humans in terms of physiology and anatomy. Microarray analysis of oocytes, before and after in vitro maturation was performed and later validated by RT-qPCR. We have particularly detected and analyzed genes belonging to gene ontology groups associated with hormonal stimulation during maturation of the oocytes, that exhibited significant change in expression (fold change ≥ |2|; p < 0.05) namely “Female sex differentiation” (CCND2, MMP14, VEGFA, FST, INHBA, NR5A1), “Response to endogenous stimulus” (INSR, ESR1, CCND2, TXNIP, TACR3, MMP14, FOS, AR, EGR2, IGFBP7, TGFBR3, BTG2, PLD1, PHIP, UBE2B) and “Response to estrogen stimulus” (INSR, ESR1, CCND2, IHH, TXNIP, TACR3, MMP14). Some of them were characteristic for just one of the described ontologies, while some belonged into multiple ontological terms. The genes were analyzed, with their relation to the processes of interest explained. Overall, the study provides us with a range of genes that might serve as molecular markers of in vitro maturation associated processes of the oocytes. This knowledge might serve as a reference for further studies and, after further validation, as a potentially useful knowledge in assessment of the oocytes during assisted reproduction processes.

Hypermethylation of the κ1 opioid receptor promoter in Chinese heroin and methamphetamine addicts.

Heroin and methamphetamine (METH) addiction continues to be a major social, economic and therapeutic problem worldwide. The opioid pathway may mediate the effects of addictive drugs. However, the potential correlation between the κ1 opioid receptor (OPRK1) and drug addiction has not yet been characterized. The aim of the present study was to investigate the potential association between methylation of the OPRK1 promoter and substance abuse. Bisulfite pyrosequencing technology was used to determine the levels of OPRK1 promoter methylation in 60 drug abusers (30 heroin and 30 METH addicts) and 52 controls, observed to exhibit no significant differences in age or gender. The results indicated that levels of OPRK1 promoter methylation were significantly higher in drug addicts when compared with controls (P=2.43×10−4). Significant correlations between OPRK1 promoter methylation and the length and frequency of drug use were also observed in male heroin addicts (length: r=0.661, P=0.007; frequency: r=−0.684, P=0.005). In addition, a luciferase reporter gene assay indicated that the OPRK1 promoter fragment was able to regulate gene expression (fold change between two groups >32.12, P≤0.0001). In conclusion, results of the present study indicate that methylation of the OPRK1 promoter contributes to the pathophysiology of drug addiction.

LncRNA and mRNA interaction study based on transcriptome profiles reveals potential core genes in the pathogenesis of human thoracic aortic dissection.

The aim of the present study was to determine the potential core genes in the pathogenesis of human thoracic aortic dissection (TAD) by analyzing microarray profiles of long non-coding (lnc)-RNAs between TAD and normal thoracic aorta (NTA). The differentially expressed lncRNA profiles of the aorta tissues between TAD patients (TAD group, n=6) and age-matched donors with aortic diseases (NTA group, n=6) were analyzed by lncRNAs microarray. Gene ontology (GO), pathway and network analyses were used to further investigate candidate lncRNAs and mRNAs. Differentially expressed lncRNAs and mRNAs were validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). In total, the present study identified 765 lncRNAs and 619 mRNAs with differential expression between TAD and NTA (fold change >2.0, P<0.01). GO analysis demonstrated that the differentially upregulated lncRNAs are associated with cell differentiation, homeostasis, cell growth and angiogenesis. Kyoto Encyclopedia of Gene and Genomes pathway analysis demonstrated that the differentially downregulated lncRNAs are mainly associated with arrhythmogenic right ventricular cardiomyopathy, hypertrophic cardiomyopathy and dilated cardiomyopathy. To reduce the lncRNAs for further investigation and to enrich those potentially involved in TAD, a total of 16 candidate lncRNAs with a significant expression (fold change >4, P<0.01) were selected, that were associated with an annotated protein-coding gene through the GO term and scientific literatures. Then a set of significantly expressed lncRNAs [purinergic receptor P2X7 (P2RX7), hypoxia inducing factor (HIF)-1A-AS2, AX746823, RP11-69I8.3 and RP11-536K7.5) and the corresponding mRNAs (P2RX7, cyclin dependent kinase inhibitor 2B, HIF-1A, runt-related transcription factor 1, connective tissue growth factor and interleukin 2 receptor a chain] were confirmed using RT-qPCR. The present study revealed that the expression profiles of lncRNAs and mRNAs in aorta tissues from TAD were significantly altered. These results may provide important insights into the pathogenesis of TAD disease.

Coexpression network revealing the plasticity and robustness of population transcriptome during the initial stage of domesticating energy crop Miscanthus lutarioriparius.

Coexpression network revealing genes with Co-variation Expression pattern (CE) and those with Top rank of Expression fold change (TE) played different roles in responding to new environment of Miscanthus lutarioriparius. Variation in gene expression level, the product of genetic and/or environmental perturbation, determines the robustness-to-plasticity spectrum of a phenotype in plants. Understanding how expression variation of plant population response to a new field is crucial to domesticate energy crops. Weighted Gene Coexpression Network Analysis (WGCNA) was used to explore the patterns of expression variation based on 72 Miscanthus lutarioriparius transcriptomes from two contrasting environments, one near the native habitat and the other in one harsh domesticating region. The 932 genes with Co-variation Expression pattern (CE) and other 932 genes with Top rank of Expression fold change (TE) were identified and the former were strongly associated with the water use efficiency (r ≥ 0.55, P ≤ 10-7). Functional enrichment of CE genes were related to three organelles, which well matched the annotation of twelve motifs identified from their conserved noncoding sequence; while TE genes were mostly related to biotic and/or abiotic stress. The expression robustness of CE genes with high genetic diversity kept relatively stable between environments while the harsh environment reduced the expression robustness of TE genes with low genetic diversity. The expression plasticity of CE genes was increased less than that of TE genes. These results suggested that expression variation of CE genes and TE genes could account for the robustness and plasticity of acclimation ability of Miscanthus, respectively. The patterns of expression variation revealed by transcriptomic network would shed newlight on breeding and domestication of energy crops.

Alternative splicing of helicase-like transcription factor (Hltf): Intron retention-dependent activation of immune tolerance at the feto-maternal interface.

Hltf is regulated by intron retention, and global Hltf-deletion causes perinatal lethality from hypoglycemia. In heart, full-length Hltf is a transcriptional regulator of Hif-1α that controls transport systems. Thus, we tested the hypothesis that Hltf deletion from placenta caused or exacerbated neonatal hypoglycemia via Hif-1α regulation of nutrient transporters. RNA-seq data analyses identified significant changes in transcript expression and alternative splicing (AS) in E18.5 placentome. iPathwayGuide was used for gene ontology (GO) analysis of biological processes, molecular functions and cellular components. Elim pruning algorithm identified hierarchical relationships. The methylome was interrogated by Methyl-MiniSeq Epiquest analysis. GO analysis identified gene enrichment within biological processes. Protein expression was visualized with immunohistochemistry. Although two Hltf mRNA isoforms are quantifiable in most murine tissues, only the truncated Hltf isoform is expressed in placenta. The responsible intron retention event occurs in the absence of DNA methylation. iPathwayGuide analysis identified 157 target genes of 11,538 total genes with measured expression. These were obtained using a threshold of 0.05 for statistical significance (p-value) and a long fold change of expression with absolute value of at least 0.6. Hltf deletion altered transcription of trophoblast lineage-specific genes, and increased transcription of the Cxcr7 (p = 0.004) gene whose protein product is a co-receptor for human and simian immunodeficiency viruses. Concomitant increased Cxcr7 protein was identified with immunolabeling. Hltf deletion had no effect on transcription or site-specific methylation patterns of Hif-1α, the major glucose transporters, or System A amino acid transporters. There was no measureable evidence of uteroplacental dysfunction or fetal compromise. iPathGuide analysis revealed Hltf suppresses cytolysis (10/21 genes; p-value 1.900e-12; p-value correction: Elim pruning; GO:019835) including the perforin-granzyme pathway in uterine natural killer cells. Our findings 1) prove the truncated Hltf protein isoform is a transcription factor, 2) establish a functional link between AS of Hltf and immunosuppression at the feto-maternal interface, 3) correlate intron retention with the absence of DNA methylation, and 4) underscore the importance of differential splicing analysis to identify Hltf’s functional diversity.

Profiles of differentially expressed circRNAs in esophageal and breast cancer.

IntroductionCircular RNAs (circRNAs) function as efficient microRNA sponges with gene-regulatory potential and are promising cancer biomarkers. In this study, we used the Arraystar Human circRNA Array to construct a genome-wide circRNA profile of esophageal squamous cell cancer (ESCC) and breast cancer (BC).Patients and methodsExpression levels between cancer lesions and adjacent normal-appearing tissues were compared. We observed 469 upregulated circRNAs and 275 downregulated circRNAs in ESCC. Hsa_circRNA_103670 was upregulated 20.3-fold, while hsa_circRNA_030162 was downregulated 12.1-fold. For BC, 715 circRNAs were upregulated, and 440 circRNAs were downregulated. Hsa_circRNA_005230 was upregulated 12.2-fold, while hsa_circRNA_406225 was downregulated 12.4-fold.ResultsWhen we set the criteria as fold change in expression ≥2 between cancer and adjacent normal-appearing tissue with a P-value <0.01, there were 22 common circRNAs (11 upregulated and 11 downregulated) in relation to both ESCC and BC. Gene ontology and the Kyoto encyclopedia of genes and genomes analyses showed that these circRNAs were involved in the tumorigenesis of human cancers.ConclusionOur study revealed that circRNAs are promising candidates as valuable biomarkers for ESCC and BC, although relevant research is still in its infancy and the functional role of specific circRNAs in tumorigenesis is just starting to be elucidated.

Abstract 1494: SOX18: A novel master regulator of high-grade serous ovarian tumorigenesis

Abstract High-grade serous ovarian cancer (HGSOC) is the most common and lethal subtype of ovarian cancer. Recent data indicate that fallopian tube secretory epithelial cells (FTSECs) represent the cell of origin for HGSOC. While many studies have characterized molecular features associated with HGSOC biology, the master transcription factors (TFs) that drive disease development are not known. To identify master TFs for HGSOC, we first performed an integrative analysis of gene expression profiles from 73 FTSEC samples and 394 HGSOCs from The Cancer Genome Atlas (TCGA) project. We then integrated these data with chromatin immunoprecipitation sequencing (ChIP-seq) analyses performed on primary HGSOC samples to select active chromatin regions marked by H3k27ac and to identify super-enhancer (SE) regions. These analyses identified overexpressed TFs that coincide with tumor-specific SEs, a hallmark of master TFs. We identified multiple putative master TFs including SOX18, which was highly overexpressed in HGSOCs relative to normal FTSECs (log2 fold change in expression = 3.5, p = 2.5x10-19). SOX18 is a TF that regulates development of blood and lymphatic vessels, but its association with HGSOC development has not been reported before. We examined the expression of SOX18 in ~8,000 tumors representing 17 tumor types from TCGA. SOX18 expression was significantly elevated in HGSOC relative to any other tumor types, suggesting it is highly specific to this cancer. We found that SOX18 is highly expressed in the HGSOC cell lines UWB1.289, Kuramochi, and EFO21, and we then performed lentiviral-mediated shRNA knockdown of SOX18 in these cell lines to establish the effects of SOX18 depletion on neoplastic phenotypes. In ongoing experiments, we are performing gene expression profiling and H3k27ac ChIP-seq analysis after SOX18 knockdown to identify SOX18 target genes and characterize the landscape of SOX18 binding sites in HGSOC. These studies will likely identify novel molecular biomarkers that may represent much-needed therapeutic targets for HGSOC. Citation Format: Annie Y. Liu, Kevin C. Vavra, Rosario I. Corona, Forough Abassi, Marcos Fonseca, Felipe Segato, Matthew L. Freedman, Simon A. Gayther, Houtan Noushmehr, Kate Lawrenson. SOX18: A novel master regulator of high-grade serous ovarian tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1494.

Abstract 3356: The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines

Abstract BACKGROUND: Ewing sarcoma (ES) is the second most common pediatric bone cancer. ES cells are dependent on the activity of the EWS/FLI1 transcription factor for cell survival. EWS/FLI1 drives proliferation, dysregulates the cell cycle and establishes a block in differentiation. Despite the known dependence of Ewing sarcoma on EWS/FLI1, the exact identity of all the downstream targets of EWS/FLI1 is not known. While gene signatures have been reported for EWS/FLI1, these fail to capture the marked heterogeneity in the EWS/FLI1 transcriptome or correlate these expression changes with cellular phenotype. METHODS: Next generation RNA sequencing with deep coverage was used to generate a comprehensive analysis of the EWS/FLI1 transcriptome by silencing EWS/FLI1 in 5 different ES cell lines at two different time points. These results were organized into gene expression networks and signaling nodes were identified by weighted correlation network analysis (WGCNA). The networks were validated by a 500-gene signature of EWS/FLI1 targets using a next generation sequencing capture assay and the Illumina platform. The cellular endpoint of EWS/FLI1 silencing was evaluated by caspase 3,7 cleavage to measure apoptosis, BGAL staining to measure senescence and scratch assays to capture migration. RESULTS: Next generation RNA sequencing with siRNA silencing of EWS/FLI1 revealed marked heterogeneity in the EWS/FLI1 transcriptome among cell lines. Targets of EWS/FLI1 that were common across cell lines exhibited small magnitude gene expression changes (log fold &amp;lt;2), while large magnitude gene expression changes were unique to individual cell lines (log fold&amp;gt;2). When the log fold change in expression was restricted to greater than 2, only 13 common repressed targets and 4 common induced targets were identified in all 5 cell lines. In addition, silencing of EWS/FLI1 induced dramatically different phenotypes across cell lines, where p53 wild-type cell lines underwent senescence and the p53 mutant cell lines did not. Most cell lines did not continue to proliferate with EWS/FLI1 silencing with the exception being the widely used A673 cell line. Cellular migration did not change with EWS/FLI1 silencing in many of the cell lines in contrast to A673 cells. CONCLUSIONS: EWS/FLI1 drives different gene expression signatures and different phenotypes in unique cell lines. This diversity needs to be considered as EWS/FLI1 directed therapies are developed. Citation Format: Susan M. Kitchen-Goosen, Megan J. Bowman, Marie Adams, Penny Berger, Patrick J. Grohar. The EWS/FLI1 transcriptome is characterized by marked heterogeneity across Ewing sarcoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3356.

Abstract C47: Obesity enhances tumor colonization and promotes expression of the spliced variant MBD2_v2 and splicing factor SRSF2 in triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is a molecular subtype that accounts for approximately 15-20% of invasive breast cancer diagnoses in the United States. TNBC occurs more prevalently in premenopausal African American women (AAW), and obesity coupled with chronic inflammation is a risk factor for both incidence and recurrence. TNBC is lacking therapeutic options and persons diagnosed with TNBC have the lowest 5-year survival rates among all breast cancer patients, with AAW disproportionally bearing this burden. In order to improve TNBC patient outcomes, etiologic factors driving disparities in disease progression need to be better understood. We previously showed in TNBC cell cultures that intrinsic oncogenic redox signaling promotes self-renewal capacity of cancer stem-like cells (CSC) by maintaining expression of an epigenetic reader protein and mRNA spliced variant, methyl binding domain protein 2 variant 2 (MBD2_v2). We hypothesized that obesity-induced chronic inflammation in vivo could promote a similar cancer phenotype. Methods: To study this potential phenomenon, we developed and characterized a diet-induced obesity mouse tumor xenograft (MTX) model of TNBC in C57BL/6J Rag1 (-/-) mice, a strain that maintains a functional innate immune system, to recapitulate the proinflammatory environment induced by increased adiposity. TNBC cell lines MDA-MB-468 and MDA-MB-231 were used to generate ectopic tumor xenografts in this model. Tumor colonization and growth were measured over 150 days. RT-PCR and microarray based assays were used to detect transcriptional changes in TNBC MTXs. Results: At 5 weeks post-diet consumption, mice fed a high-fat diet displayed a 5-10% increase in body weight and increased blood glucose concentration. By the end of the study high-fat fed mice displayed a 33-43% increase in body weight and appeared to have greater central adiposity post-mortem. In overweight mice relative to healthy weight controls, TNBC cell line tumor colonization rate and MBD2_v2 expression were significantly higher. Furthermore, staining for CSC marker EpCam was greater in overweight versus control MTXs, indicating that CSC self-renewal capacity could have been enhanced. Upon global gene expression analysis of overweight MTXs, we observed a significant upregulation of stem-related factors based on fold-change expression, as well as functional enrichment for RNA splicing, including SRSF2, a splicing factor that has been shown to drive MBD2_v2 expression in pluripotent stem cells. Knockdown of SRSF2 led to decreased expression of MBD2_v2 in MDA-MB-468 cells, and decreased CSC self-renewal capacity in vitro. Conclusions: Altogether these data support MBD2_v2 regulation by redox signaling, which may be driven by a proinflammatory environment in the presence of increased adiposity. Future studies will be conducted to confirm MBD2_v2's importance in obesity-related tumor colonization in in vivo overexpression and knockdown studies, and to determine its transcriptional regulatory functions in TNBC. Citation Format: Emily A. Girsch, Gregory Dyson, Cristina Mitrea, Bin Bao, Lisa Polin, Aliccia Bollig-Fischer. Obesity enhances tumor colonization and promotes expression of the spliced variant MBD2_v2 and splicing factor SRSF2 in triple-negative breast cancer [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr C47.

Molecular profiling of postreperfusion milieu determines acute kidney injury after liver transplantation: A prospective study.

Acute kidney injury (AKI) after liver transplantation (LT) is a common event, but its pathogenesis remains unclear. The aim of this prospective study is to investigate the potential relationship between postreperfusion gene expression, serum mediators, and the onset of AKI after LT. Sixty-five consecutive patients undergoing LT were included in the study. Reverse transcription polymerase chain reaction (PCR) was performed on liver biopsies. Gene expression of 23 genes involved in ischemia/reperfusion injury (IRI) was evaluated. The serum concentrations of endothelin (ET)-1 and inflammatory cytokines were analyzed. AKI after LT developed in 21 (32%) recipients (AKI group). Reverse transcription PCR of reperfusion biopsy in the AKI group showed higher expression of several genes involved in IRI compared with the non-AKI group. Fold changes in the gene expression of ET-1, interleukin (IL) 18, and tumor necrosis factor α (TNF-α) were associated with creatinine peak value. AKI patients also had significantly higher ET-1, IL18, and TNF-α postoperative serum levels. Multivariate analysis showed that ET-1 (odds ratio [OR], 16.7; 95% confidence interval [CI], 3.34-83.42; P = 0.001) and IL18 (OR, 5.27; 95% CI, 0.99-27.82, P = 0.048) serum levels on postoperative day 1 were independently predictive of AKI. Receiver operating characteristic analysis demonstrated that the combination of biomarkers ET-1+IL18 was highly predictive of AKI (area under the receiver operating characteristic curve, 0.91; 95% CI, 0.83-0.99). Early allograft dysfunction and chronic kidney disease stage ≥ 2 occurred more frequently in AKI patients. These results suggest that the graft itself, rather than intraoperative hemodynamic instability, plays a main role in AKI after LT. These data may have mechanistic and diagnostic implications for AKI after LT. Liver Transplantation 24 922-931 2018 AASLD.

PO-349 Identification of differentially expressed lncRNAs in metformin resistant SH-SY5Y neuroblastoma cells

IntroductionOf the total human DNA only 1.2% o codes for proteins, with the rest only being transcribed into regulatory RNA, known as non-coding RNA. Long non-coding RNAs (lncRNAs) are non-coding RNAs that have no or limited protein-coding potential, are >200 nt in length and include- among other categories- long/large intergenic/intervening RNAs (lincRNAs), intronic lncRNAs, natural antisense transcripts (NATs) and pseudogenes. lncRNAs are key regulators of several cellular processes and have been associated with a variety of diseases, including cancer. Metformin (N, N-dimethyl biguanide) is an oral biguanide already in clinical use against diabetes and has also been suggested to decrease the incidence of various cancers, including neuroblastoma, the most common extracranial paediatric cancer arising at the sympathetic nervous system.Material and methodsThe present study aimed at assessing the differential expression of protein-coding and, primarily, non-protein-coding RNAs between control untreated SH-SY5Y neuroblastoma cells and cells resistant to 3 mM Metformin via a paired-end RNA sequencing approach.Results and discussionsIn the first data set (untreated cells compared to cells resistant to 3 mM Metformin), out of 13 741 genes measured, 7855 genes were found to be differentially expressed with 108 of them falling within the lncRNA category. Among these 33 comprise lincRNAs, 24 are NATs and 18 pseudogenes. In the second data set (untreated cells compared to cells treated with 20 mM Metformin) 13 481 genes were tested, of which 5652 showed perturbed expression. Among them, 86 belong to the lncRNA category, and in particular, 33 are lincRNAs, 19 are NATs and 13 are pseudogenes. Interestingly, 34 of the assessed lncRNAs display differential expression in both data sets. The results were obtained using a threshold of 0.05 for statistical significance (p-value) and a log fold change of expression with an absolute value of at least 0.6.Neuroblastoma cells’ response to Metformin, as well as the acquisition of resistance to the drug, trigger the differential expression of a great diversity of lncRNAs.ConclusionGiven that Metformin is an appealing and promising therapeutic approach against neuroblastoma, these lncRNAs could, in turn, be used as molecular biomarkers towards better prediction, prognosis and diagnosis of the disease. Moreover, such an approach would be of interest as part of a combination therapy.

Abstract 2673: Securidaca1 and 2 induced apoptosis: A PI3K-AKT dependent pathway in cervical cancer

Abstract Background: Securidaca1 and 2 are plant-derived substances isolated from African medicinal plant S. longipedunculata, using new bioactivity-guided method partly described in Obasi et al. (2017). Our previous report suggested that certain fractions from this plant root were able to activate apoptosis in cervical cancer cell lines. Generally, over 90% of cervical cancer has been associated with high-risk human papilloma virus (HPV), whose etiologic molecule E6 oncoprotein is involved in the process of carcinogenesis. The purpose of this study, therefore, is to investigate the proapoptotic potentials of these substances and their ability to block cell survival mechanism, via antiapoptotic effectors of PI3K-AKT downstream. Experimental Procedure: Two cervical cancer cell lines (Caski and BU25TK), positive with HPV were used for the investigation. Cells were treated with the plant-derived substances and the IC50 was determined by MTT assay. Fluorescence microscopy (Annexin V-FITC, MPC and PI staining) was used to investigate autophagy/apoptosis, while relevant genes were analyzed by RT-qPCR. Results: Reduced cell proliferation was earlier achieved in dose- and time-dependent manner by fractions retaining Securidaca1 and 2, with IC50 of 7.03 and 16.39 (μg/mL) respectively on Caski cells. Late apoptosis was also activated on cells treated with securidaca1 fraction, while the securidaca2 in contrast indicated early apoptosis. Analysis by RT-qPCR revealed a fold-change expression of antiapoptotic proteins, MCL-1 and BCL2L1, more pronounced in securidaca1. Interestingly, the AKT-3 was significantly inhibited, correlating with low expression of MCL-1, BCL2L1, VEGFA and CDH-1. Data analysis suggests the most likely activation of apoptosis via PTEN regulated PI3K-AKT pathway and respondent downstream (mTOR/ NF-kB). Conclusion: The ability of Securidaca1 and 2 to kill tumor cells and induce apoptosis in manner involving multiple regulatory pathways enhances their potential against possible resistance and makes them attractive as candidates for anticancer development. Citation Format: Titus C. Obasi, Cornelia Braicu, Heiko Noack, Ioana Berindan-Neagoe, Bjorn Junker, Radu N. Oprean. Securidaca1 and 2 induced apoptosis: A PI3K-AKT dependent pathway in cervical cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2673.

InDePTH: detection of hub genes for developing gene expression networks under anticancer drug treatment.

It has been difficult to elucidate the structure of gene regulatory networks under anticancer drug treatment. Here, we developed an algorithm to highlight the hub genes that play a major role in creating the upstream and downstream relationships within a given set of differentially expressed genes. The directionality of the relationships between genes was defined using information from comprehensive collections of transcriptome profiles after gene knockdown and overexpression. As expected, among the drug-perturbed genes, our algorithm tended to derive plausible hub genes, such as transcription factors. Our validation experiments successfully showed the anticipated activity of certain hub gene in establishing the gene regulatory network that was associated with cell growth inhibition. Notably, giving such top priority to the hub gene was not achieved by ranking fold change in expression and by the conventional gene set enrichment analysis of drug-induced transcriptome data. Thus, our data-driven approach can facilitate to understand drug-induced gene regulatory networks for finding potential functional genes.

Platelet rich fibrin sealant Vivostat ® in pancreatic surgery

The processes underlying maturation of mammalian oocytes are considered crucial for the oocytes ability to undergo monospermic fertilization. The same factors of influence are suggested to impact the development of sex associated characteristics, allowing sex differentiation to progress during embryonic growth. The primary aim of the study was to analyze the gene ontology groups involved in regulation of porcine oocytes' response to endogenous stimuli. The results obtained would indicate potential genes influencing sex differentiation. Additionally, they could help to determine new genetic markers, expression profile of which is substantially regulated during porcine oocytes’ in vitro maturation. To achieve that, porcine oocytes were collected for analysis before and after in vitro maturation. Pigs were used as they are a readily available model that presents significant similarity to humans in terms of physiology and anatomy. Microarray analysis of oocytes, before and after in vitro maturation was performed and later validated by RT-qPCR. We have particularly detected and analyzed genes belonging to gene ontology groups associated with hormonal stimulation during maturation of the oocytes, that exhibited significant change in expression (fold change ≥ |2|; p < 0.05) namely “Female sex differentiation” (CCND2, MMP14, VEGFA, FST, INHBA, NR5A1), “Response to endogenous stimulus” (INSR, ESR1, CCND2, TXNIP, TACR3, MMP14, FOS, AR, EGR2, IGFBP7, TGFBR3, BTG2, PLD1, PHIP, UBE2B) and “Response to estrogen stimulus” (INSR, ESR1, CCND2, IHH, TXNIP, TACR3, MMP14). Some of them were characteristic for just one of the described ontologies, while some belonged into multiple ontological terms. The genes were analyzed, with their relation to the processes of interest explained. Overall, the study provides us with a range of genes that might serve as molecular markers of in vitro maturation associated processes of the oocytes. This knowledge might serve as a reference for further studies and, after further validation, as a potentially useful knowledge in assessment of the oocytes during assisted reproduction processes.