Organismal Injury Research Articles

Melatonin Promotes Cheliped Regeneration, Digestive Enzyme Function, and Immunity Following Autotomy in the Chinese Mitten Crab, Eriocheir sinensis.

In the pond culture of juvenile Eriocheir sinensis, a high limb-impairment rate seriously affects the culture success. Therefore, it is particularly important to artificially promote limb regeneration. This study evaluated the effects of melatonin on cheliped regeneration, digestive ability, and immunity, as well as its relationship with the eyestalk. It was found that the injection of melatonin significantly increased the limb regeneration rate compared with the saline group (P < 0.05). The qRT-PCR results of growth-related genes showed that the level of EcR-mRNA (ecdysteroid receptor) and Chi-mRNA (chitinase) expression was significantly increased following the melatonin injection, while the expression of MIH-mRNA (molt-inhibiting hormone) was significantly decreased (P < 0.05). Melatonin significantly increased lipase activity (P < 0.05). We observed that the survival rates of limb-impaired and unilateral eyestalk-ablated crabs were substantially improved following melatonin treatment, whereas the survival of the unilateral eyestalk-ablated crabs was significantly decreased compared with the control group (P < 0.05). Furthermore, the results of serum immune and antioxidant capacity revealed that melatonin significantly increased the total hemocyte counts (THC), hemocyanin content, total antioxidant capacity (T-AOC), acid phosphatase (ACP), and glutathione peroxidase activity (GSH-Px), whereas the immune-related parameters were significantly decreased in eyestalk-ablated crabs (P < 0.05). Therefore, these findings indicate that melatonin exerts a protective effect on organism injury, which could promote limb regeneration by up-regulating the expression of growth-related genes, improve digestive enzyme activity, and strengthen the immune response, particularly antioxidant capacity.

Abstract 77: Circulating miRNome in Moyamoya Discordant MZ Twins - Implications for Endothelial Biofunctions

Objective: We aim to investigate circulating genome-wide microRNA (miRNome) profiles in Moyamoya disease-discordant monozygotic (MZ) twins with the RNF213 founder mutation (rs112735431), since the etiology remains unclear even after the identification of RNF213. Methods: Circulating miRNome was screened in a pair of Moyamoya-discordant MZ twins and a validation cohort (unrelated 9 cases and 8 normal controls) using microarray and validated by quantitative real-time PCR. Differential plasma microRNAs were quantified in endothelial cells differentiated from iPS cell line (iPSECs) derived from unrelated 3 cases and 3 controls. Gene expressions targeted by the significant microRNAs in iPSECs was studied using published iPSEC-transcriptome and bioinformatics tool. Results: A total of 537 circulating microRNAs were detected in the Moyamoya-discordant MZ twins and the validation cohort. Unsupervised cluster analysis demonstrated 2 discrete miRNome, predominantly by disease, but the MZ twins showed similar miRNome profiles, regardless of phenotypic discordance (Fig. 1A). Across the 143 and 98 differential microRNAs in the MZ twins and validation cohort, 23 microRNAs were overlapped (Fig. 1B). A cluster of plasma hsa-miR-6722-3p/-328-3p/150-5p was significantly up-regulated in patients in the validation cohort (p &lt; 0.05) (Fig. 1C). In iPSECs, hsa-miR-6722-3p/-328-3p cluster was up-regulated with a greater than 3.0-fold change in patients. Bioinformatics analysis revealed that 94 target genes involving organismal injuries and cell survival/maintenance were significantly down-regulated in Moyamoya-iPSECs (p &lt; 0.05, q &lt; 0.1), suggesting the regulatory role of the miR-6722/-328 cluster in endothelial gene expression (Fig. 1D). Conclusions: Differential plasma-miRNAs in Moyamoya-discordant MZ twins may potentially serve as a diagnostic biomarker in Moyamoya disease and regulate endothelial gene expression.

Perturbation of microRNA signalling by doxorubicin in spermatogonial, Leydig and Sertoli cell lines in vitro.

We have previously shown that in addition to its widely recognised cardiotoxicity, the chemotherapeutic doxorubicin (DOX) is able to induce transcriptional, microRNA (miRNA) and DNA methylation changes in the mouse testis. These changes perturb pathways involved in stress/cell death and survival and testicular function and lead to germ cell loss and reproductive organ damage. Here, we further investigated the differential miRNA expression induced by DOX in mouse spermatogonial (GC1), Leydig (TM3) and Sertoli (TM4) cell lines in vitro. We began by performing cell cycle analysis of the three mouse testicular cell lines to evaluate their sensitivity to DOX and thus select suitable doses for miRNA profiling. In keeping with our in vivo data, the spermatogonial cell line was the most sensitive, and the Sertoli cell line the most resistant to DOX-induced cell cycle arrest. We then further demonstrated that each cell line has a distinct miRNA profile, which is perturbed upon treatment with DOX. Pathway analysis identified changes in the miRNA-mediated regulation of specialised signalling at germ-Sertoli and Sertoli-Sertoli cell junctions following treatment with DOX. Amongst the most significant disease categories associated with DOX-induced miRNA expression were organismal injury and abnormalities, and reproductive system disease. This suggests that miRNAs play significant roles in both normal testicular function and DOX-induced testicular toxicity. Comparison of our in vitro and in vivo data highlights that in vitro cell models can provide valuable mechanistic information, which may also help facilitate the development of biomarkers of testicular toxicity and high-throughput in vitro screening methods to identify potential testicular toxicants.

Chronic exposure to acephate triggers ROS-mediated injuries at organismal and sub-organismal levels of Drosophila melanogaster.

The present study demonstrates ROS-mediated organismal and sub-organismal injuries in Drosophila melanogaster following chronic acephate exposure. Larvae and adults of Drosophila were reared on food supplemented with sub-lethal concentrations (1-6 μg mL-1) of acephate (LC50 8.71 μg mL-1). The longevity of the treated adults was reduced to half at 6 μg mL-1 exposure along with declined neuromuscular coordination and physical activities. Apparent developmental defects in the compound eyes were confirmed through the detection of apoptotic lesions in larval eye imaginal discs. The larval gut manifested tissue damage at various sites. Neural and fat cell viability was reduced by ∼1.89- and ∼3.38-fold at 6 μg mL-1 acephate treatment, respectively. A significant reduction in hemocyte viability confirmed the immunotoxic potential of acephate. Nearly 1-3-fold enhancement in the expression of OS markers (MDA, protein carbonyl contents, SOD, catalase and HSP70) in the treated larvae served as evidence of ROS production. The post-treatment increase in CYP450 and GST activities reflects the 'switch-on' states of the phase-I and phase-II detoxification mechanism. The genotoxic potential of acephate was confirmed through alkaline single cell gel electrophoresis. Thus, the findings of the present study validate the fact that besides traditional cholinesterase inhibition, chronic sub-lethal exposure to acephate potentially induces ROS-mediated toxic responses in Drosophila.

Systematic integrative analysis of gene expression identifies HNF4A as the central gene in pathogenesis of non-alcoholic steatohepatitis.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, and encompasses a spectrum from simple steatosis to steatohepatitis (NASH). There is currently no approved pharmacologic therapy against NASH, partly due to an incomplete understanding of its molecular basis. The goal of this study was to determine the key differentially expressed genes (DEGs), as well as those genes and pathways central to its pathogenesis. We performed an integrative computational analysis of publicly available gene expression data in NASH from GEO (GSE17470, GSE24807, GSE37031, GSE89632). The DEGs were identified using GEOquery, and only the genes present in at least three of the studies, to a total of 190 DEGs, were considered for further analyses. The pathways, networks, molecular interactions, functional analyses were generated through the use of Ingenuity Pathway Analysis (IPA). For selected networks, we computed the centrality using igraph package in R. Among the statistically significant predicted networks (p-val < 0.05), three were of most biological interest: the first is involved in antimicrobial response, inflammatory response and immunological disease, the second in cancer, organismal injury and development and the third in metabolic diseases. We discovered that HNF4A is the central gene in the network of NASH connected to metabolic diseases and that it regulates HNF1A, an additional transcription regulator also involved in lipid metabolism. Therefore, we show, for the first time to our knowledge, that HNF4A is central to the pathogenesis of NASH. This adds to previous literature demonstrating that HNF4A regulates the transcription of genes involved in the progression of NAFLD, and that HNF4A genetic variants play a potential role in NASH progression.

Comprehensive Expression Analysis of microRNAs and mRNAs in Synovial Tissue from a Mouse Model of Early Post-Traumatic Osteoarthritis

To better understand the molecular processes involved in driving osteoarthritis disease progression we characterized expression profiles of microRNAs (miRNA) and mRNAs in synovial tissue from a post-traumatic OA mouse model. OA was induced in 10–12 week old male C57BL6 mice by bilateral surgical destabilization of the medial meniscus (DMM). RNA isolated from the anterior synovium of mice at 1 and 6 weeks post-surgery was subject to expression profiling using Agilent microarrays and qPCR. OA severity was determined histologically. Anterior and posterior synovitis decreased with post-operative time after sham and DMM. No differences in synovitis parameters were evident between sham and DMM in the anterior synovium at either time. While expression profiling revealed 394 miRNAs were dysregulated between 1 and 6 week time-points in the anterior synovium, there were no significant changes in miRNA or mRNA expression between DMM and sham mice at both time-points. Bioinformatic analysis of the miRNAs and mRNAs differentially expressed in tandem with the resolution of anterior synovial inflammation revealed similar biological processes and functions, including organismal injury, connective tissue disorder and inflammatory responses. Our data demonstrates that early OA-specific patterns of synovial miRNAs or mRNAs dysregulation could not be identified in this model of post-traumatic OA.

Knockdown of TBRG4 affects tumorigenesis in human H1299 lung cancer cells by regulating DDIT3, CAV1 and RRM2.

The transforming growth factor β regulator 4 (TBRG4) gene, located on the 7p14-p13 chromosomal region, is implicated in numerous types of cancer. However, the contribution(s) of TBRG4 in human lung cancer remains unknown. In the present study, the expression of TBRG4 mRNA was investigated in the H1299 lung cancer cell line using the quantitative polymerase chain reaction (qPCR) following the knockdown of TBRG4 by a lentivirus-mediated small interfering RNA (siRNA). Results identified that the expression of TBRG4 within H1299 cells was significantly suppressed (P<0.01) by RNA interference, and 586 genes were differentially expressed following TBRG4 silencing. Ingenuity Pathway Analysis (IPA) revealed that these genes were often associated with infectious diseases, organismal injury, abnormalities and cancer functional networks. Further IPA of these networks revealed that TBRG4 knockdown in H1299 cells deregulated the expression of 21 downstream genes, including the upregulation of DNA damage-inducible transcript 3 (DDIT3), also termed CCAAT/enhancer-binding protein homologous protein, and downregulation of caveolin 1 (CAV1) and ribonucleotide reductase regulatory subunit M2 (RRM2). Results were validated using qPCR and western blotting. Furthermore, immunohistochemical staining of TBRG4 protein identified that expression was markedly increased in carcinoma compared with in normal tissue. In conclusion, TBRG4 serves a role in the tumorigenesis of lung cancer via deregulation of DDIT3, CAV1 and RRM2. The results of the present study may be important in contributing to our understanding of TBRG4 as a target for lung cancer treatment.

Knockdown of Transforming Growth Factor Beta Regulator 4 (Tbrg4) Promotes Apoptosis in Human Lung Cancer Cell Line H1299 via Regulating Ddit3, Cav1 and Rrm2

Abstract Transforming growth factor beta regulator 4 (TBRG4) gene is located on 7 p14-p13, a region correlated with many cancer types, such as hematologic malignancies, head and neck squamous cell carcinoma, and multiple myeloma. However, the role of TBRG4 in human lung cancer is largely unknown. Thus, in the present study, the TBRG4 mRNA level was evaluated in the lung cancer cell line H1299 by quantitative PCR (qPCR) after knocking down TBRG4 using lentivirus-mediated small interfering RNA (siRNA). The result showed that TBRG4 was significantly inhibited in H1299 by RNAi. Microarray analysis revealed 586 differentially-expressed genes after TBRG4 silencing. Analysis of the dataset with differentially-expressed genes using Ingenuity Pathway Analysis (IPA) software showed that infectious diseases, cancer, organismal injury and abnormalities were functions associated with the highest rated network. Further analysis of this network by IPA revealed that TBRG4 knockdown in H1299 cells changed the levels of downstream genes, including the upregulation of DDIT3 and downregulation of CAV1 and RRM2. These results have been validated by qPCR and Western blotting. Moreover, TBRG4 was shown to be highly expressed in carcinoma by immunohistochemstry compared with adjacent carcinomatous normal tissue. However, there were no associations between TBRG4 expression and clinicopathological characteristic, including sex, age, distant metastasis, TNM stage, and tumour grade. Taken together, TBRG4 plays an anti-apoptotic role in tumorigenesis of lung cancer via regulating DDIT3, CAV1 and RRM2 expression. The present findings are potentially significant to advance the understanding of TBRG4 as a candidate for the treatment of lung cancer.

Protein levels of clusterin and glutathionesynthetase in platelets allow for early detection of colorectal cancer.

Colorectal cancer (CRC) is one of the most frequent malignancies in the Western world. Early tumor detection and intervention are important determinants on CRC patient survival. During early tumor proliferation, dissemination and angiogenesis, platelets store and segregate proteins actively and selectively. Hence, the platelet proteome is a potential source of biomarkers denoting early malignancy. By comparing protein profiles of platelets between healthy volunteers (n=12) and patients with early- (n=7) and late-stage (n=5) CRCs using multiplex fluorescence two-dimensional gel electrophoresis (2D-DIGE), we aimed at identifying differentially regulated proteins within platelets. By inter-group comparisons, 94 differentially expressed protein spots were detected (p<0.05) between healthy controls and patients with early- and late-stage CRCs and revealed distinct separations between all three groups in principal component analyses. 54 proteins of interest were identified by massspectrometry and resulted in high-ranked Ingenuity Pathway Analysis networks associated with Cellular function and maintenance, Cellular assembly and organization, Developmental disorder and Organismal injury and abnormalities (p<0.0001 to p=0.0495). Target proteins were validated by multiplex fluorescence-based Western blot analyses using an additional, independent cohort of platelet protein samples [healthy controls (n=15), early-stageCRCs(n=15), late-stage CRCs (n=15)]. Two proteins-clusterin and glutathionesynthetase (GSH-S)-featured high impact and were subsequently validated in this independent clinical cohort distinguishing healthy controls from patients with early- and late-stage CRCs. Thus, the potential of clusterin and GSH-S as platelet biomarkers for early detection of CRC could improve existing screening modalities in clinical application and should be confirmed in a prospective multicenter trial.

Low-dose exposure to bisphenols A, F and S of human primary adipocyte impacts coding and non-coding RNA profiles.

Bisphenol A (BPA) exposure has been suspected to be associated with deleterious effects on health including obesity and metabolically-linked diseases. Although bisphenols F (BPF) and S (BPS) are BPA structural analogs commonly used in many marketed products as a replacement for BPA, only sparse toxicological data are available yet. Our objective was to comprehensively characterize bisphenols gene targets in a human primary adipocyte model, in order to determine whether they may induce cellular dysfunction, using chronic exposure at two concentrations: a “low-dose” similar to the dose usually encountered in human biological fluids and a higher dose. Therefore, BPA, BPF and BPS have been added at 10 nM or 10 μM during the differentiation of human primary adipocytes from subcutaneous fat of three non-diabetic Caucasian female patients. Gene expression (mRNA/lncRNA) arrays and microRNA arrays, have been used to assess coding and non-coding RNA changes. We detected significantly deregulated mRNA/lncRNA and miRNA at low and high doses. Enrichment in “cancer” and “organismal injury and abnormalities” related pathways was found in response to the three products. Some long intergenic non-coding RNAs and small nucleolar RNAs were differentially expressed suggesting that bisphenols may also activate multiple cellular processes and epigenetic modifications. The analysis of upstream regulators of deregulated genes highlighted hormones or hormone-like chemicals suggesting that BPS and BPF can be suspected to interfere, just like BPA, with hormonal regulation and have to be considered as endocrine disruptors. All these results suggest that as BPA, its substitutes BPS and BPF should be used with the same restrictions.

Analysis of caspase-1 regulated transcriptome in various tissues lead to identification of novel IL-1β, IL-18 and sirtuin-1 intendent pathways

Abstract Caspase-1 is prominently involved in bridging disease risk factors/ danger signals to inflammatory response via its downstream targets such as IL (interleukin)-1β, IL-18, and Sirt-1 (Sirtuin-1). The microarray datasets derived from caspase-1 knockout tissues indicated that caspase-1 function can significantly impact the transcriptome. However, it is not known whether all effects exerted by caspase-1 on transcriptome are mediated only by its well-known substrates. Therefore, we hypothesized that the effects of caspase-1 on transcriptome may be partially independent from IL-1β, IL-18, and Sirt-1. To determine new global and tissue-specific gene regulatory effects of caspase-1, we took novel microarray data analysis approaches including Venn analysis, cooperation analysis, and meta-analysis. We made the following important findings: (1) Caspase-1 exerts its regulatory effects on majority of genes in a tissue-specific manner; (2) Caspase-1 regulatory genes partially cooperates with genes regulated by Sirt-1 during organ injury and inflammation in adipose tissue but not in liver; (3) Caspase-1 cooperates with IL-1β in regulating less than half of the genes involved in organismal injury, and cancer in mouse liver; (4) The meta-analysis identified 40 caspase-1 globally regulated genes across tissues, suggesting that caspase-1 globally regulates many novel pathways; and (5) The meta-analysis identified new cooperatively and non-cooperatively regulated genes in caspase-1, IL-1β, IL-18, and Sirt-1 pathways. Our findings suggest that caspase-1 regulates many new signaling pathways potentially via its known substrates and also via transcription factors and other proteins that are yet to be identified.

Generation and Comprehensive Analysis of Host Cell Interactome of the PA Protein of the Highly Pathogenic H5N1 Avian Influenza Virus in Mammalian Cells.

Accumulating data have identified the important roles of PA protein in replication and pathogenicity of influenza A virus (IAV). Identification of host factors that interact with the PA protein may accelerate our understanding of IAV pathogenesis. In this study, using immunoprecipitation assay combined with liquid chromatography-tandem mass spectrometry, we identified 278 human cellular proteins that might interact with PA of H5N1 IAV. Gene Ontology annotation revealed that the identified proteins are highly associated with viral translation and replication. Further KEGG pathway analysis of the interactome profile highlighted cellular pathways associated with translation, infectious disease, and signal transduction. In addition, Diseases and Functions analysis suggested that these cellular proteins are highly related with Organismal Injury and Abnormalities and Cell Death and Survival. Moreover, two cellular proteins (nucleolin and eukaryotic translation elongation factor 1-alpha 1) identified both in this study and others were further validated to interact with PA using co-immunoprecipitation and co-localization assays. Therefore, this study presented the interactome data of H5N1 IAV PA protein in human cells which may provide novel cellular target proteins for elucidating the potential molecular functions of PA in regulating the lifecycle of IAV in human cells.

DNA methylation profiling in peripheral lung tissues of smokers and patients with COPD

BackgroundEpigenetics changes have been shown to be affected by cigarette smoking. Cigarette smoke (CS)-mediated DNA methylation can potentially affect several cellular and pathophysiological processes, acute exacerbations, and comorbidity in the lungs of patients with chronic obstructive pulmonary disease (COPD). We sought to determine whether genome-wide lung DNA methylation profiles of smokers and patients with COPD were significantly different from non-smokers. We isolated DNA from parenchymal lung tissues of patients including eight lifelong non-smokers, eight current smokers, and eight patients with COPD and analyzed the samples using Illumina’s Infinium HumanMethylation450 BeadChip.ResultsOur data revealed that the differentially methylated genes were related to top canonical pathways (e.g., G beta gamma signaling, mechanisms of cancer, and nNOS signaling in neurons), disease and disorders (organismal injury and abnormalities, cancer, and respiratory disease), and molecular and cellular functions (cell death and survival, cellular assembly and organization, cellular function and maintenance) in patients with COPD. The genome-wide DNA methylation analysis identified suggestive genes, such as NOS1AP, TNFAIP2, BID, GABRB1, ATXN7, and THOC7 with DNA methylation changes in COPD lung tissues that were further validated by pyrosequencing. Pyrosequencing validation confirmed hyper-methylation in smokers and patients with COPD as compared to non-smokers. However, we did not detect significant differences in DNA methylation for TNFAIP2, ATXN7, and THOC7 genes in smokers and COPD groups despite the changes observed in the genome-wide analysis.ConclusionsOur study suggests that DNA methylation in suggestive genes, such as NOS1AP, BID, and GABRB1 may be used as epigenetic signatures in smokers and patients with COPD if the same is validated in a larger cohort. Future studies are required to correlate DNA methylation status with transcriptomics of selective genes identified in this study and elucidate their role and involvement in the progression of COPD and its exacerbations.

Activation of gga-miR-155 by reticuloendotheliosis virus T strain and its contribution to transformation.

The v-rel oncoprotein encoded by reticuloendotheliosis virus T strain (Rev-T) is a member of the rel/NF-κB family of transcription factors capable of transformation of primary chicken spleen and bone marrow cells. Rapid transformation of avian haematopoietic cells by v-rel occurs through a process of deregulation of multiple protein-encoding genes through its direct effect on their promoters. More recently, upregulation of oncogenic miR-155 and its precursor pre-miR-155 was demonstrated in both Rev-T-infected chicken embryo fibroblast cultures and Rev-T-induced B-cell lymphomas. Through electrophoresis mobility shift assay and reporter analysis on the gga-miR-155 promoter, we showed that the v-rel-induced miR-155 overexpression occurred by the direct binding to one of the putative NF-κB binding sites. Using the v-rel-induced transformation model on chicken embryonic splenocyte cultures, we could demonstrate a dynamic increase in miR-155 levels during the transformation. Transcriptome profiles of lymphoid cells transformed by v-rel showed upregulation of miR-155 accompanied by downregulation of a number of putative miR-155 targets such as Pu.1 and CEBPβ. We also showed that v-rel could rescue the suppression of miR-155 expression observed in Marek’s disease virus (MDV)-transformed cell lines, where its functional viral homologue MDV-miR-M4 is overexpressed. Demonstration of gene expression changes affecting major molecular pathways, including organismal injury and cancer in avian macrophages transfected with synthetic mature miR-155, underlines its potential direct role in transformation. Our study suggests that v-rel-induced transformation involves a complex set of events mediated by the direct activation of NF-κB targets, together with inhibitory effects on microRNA targets.

Characterization of Dysregulated miRNA in Peripheral Blood Mononuclear Cells from Ischemic Stroke Patients

Epigenetic modification may play an important role in pathophysiology of ischemic stroke (IS) risk. MicroRNAs (miRNAs), which constitute one of the modes of epigenetic regulation, have been shown to be associated with a number of clinical disorders including IS. The purpose of this study was to investigate the miRNA profile in the peripheral blood mononuclear cells (PBMCs) of IS patients and compare it with stroke-free controls. Blood samples were obtained from 19 healthy age-gender-race matched individuals who served as controls to 20 IS patients. miRNA microarray analysis with RNA from PBMCs was performed and significantly dysregulated miRNAs common among IS patients were identified. We identified 117 miRNAs with linear fold values of at least ±1.5, of which, 29 were significantly altered (p value <0.05). Ingenuity Pathway Analysis (IPA) indicated a role for the dysregulated miRNAs in conditions relevant to IS (e.g., organismal injury and abnormalities, hematological disease and immunological disease). Pro-inflammatory genes like STAT3, interleukin (IL) 12A, and IL12B were some of the highly predicted targets for the dysregulated miRNAs. Notably, we further identified three common and significantly upregulated miRNAs (hsa-miR-4656, -432, -503) and one downregulated miRNA (hsa-miR-874) among all IS patients. Molecular interactive network analysis revealed that the commonly dysregulated miRNAs share several targets with roles relevant to IS. Altogether, we report dysregulation of miRNAs in IS PBMCs and provide evidence for their involvement in the immune system alteration during IS pathophysiology.

Centromere protein U is a potential target for gene therapy of human bladder cancer.

To investigate the role of centromere protein U (CENPU) in human bladder cancer (BCa), CENPU gene expression was evaluated in human BCa tissues. We used real-time quantitative PCR (qPCR) and found that CENPU gene expression in human BCa tissues was higher compared to that observed in cancer-adjacent normal tissues. High CENPU expression was found to be strongly correlated with tumor size and TNM stage. Kaplan-Meier survival analysis indicated that high CENPU levels were associated with reduced survival. We used a lentivirus to silence endogenous CENPU gene expression in the BCa T24 cell line. CENPU knockdown was confirmed by qPCR. Cellomic imaging and BrdU assays showed that cell proliferation was significantly reduced in the CENPU-silenced cells compared to that noted in the control cells. Flow cytometry revealed that in the CENPU-silenced cells the cell cycle was arrested at the G1 phase relative to that in the control cells. In addition, apoptosis was significantly increased in the CENPU-silenced cells. Giemsa staining showed that CENPU-silenced cells, compared to control cells, displayed a significantly lower number of cell colonies. The genome-wide effect of CENPU knockdown showed that a total of 1,274 differentially expressed genes was found, including 809 downregulated genes and 465 upregulated genes. Network analysis by Ingenuity Pathway Analysis (IPA) resulted in 25 distinct signaling pathways, including the top-ranked network: ‘Cellular compromise, organismal injury and abnormalities, skeletal and muscular disorders’. In-depth IPA analysis revealed that CENPU was associated with the HMGB1 signaling pathway. qPCR and western blot analysis demonstrated that in the HMGB1 signaling pathway, CENPU knockdown downregulated expression levels of ILB, CXCL8, RAC1 and IL1A. In conclusion, our data may provide a potential pathway signature for therapeutic targets with which to treat BCa.

RNA sequencing for global gene expression associated with muscle growth in a single male modern broiler line compared to a foundational Barred Plymouth Rock chicken line

BackgroundModern broiler chickens exhibit very rapid growth and high feed efficiency compared to unselected chicken breeds. The improved production efficiency in modern broiler chickens was achieved by the intensive genetic selection for meat production. This study was designed to investigate the genetic alterations accumulated in modern broiler breeder lines during selective breeding conducted over several decades.MethodsTo identify genes important in determining muscle growth and feed efficiency in broilers, RNA sequencing (RNAseq) was conducted with breast muscle in modern pedigree male (PeM) broilers (n = 6 per group), and with an unselected foundation broiler line (Barred Plymouth Rock; BPR). The RNAseq analysis was carried out using Ilumina Hiseq (2 x 100 bp paired end read) and raw reads were assembled with the galgal4 reference chicken genome. With normalized RPM values, genes showing >10 average read counts were chosen and genes showing <0.05 p-value and >1.3 fold change were considered as differentially expressed (DE) between PeM and BPR. DE genes were subjected to Ingenuity Pathway Analysis (IPA) for bioinformatic functional interpretation.ResultsThe results indicate that 2,464 DE genes were identified in the comparison between PeM and BPR. Interestingly, the expression of genes encoding mitochondrial proteins in chicken are significantly biased towards the BPR group, suggesting a lowered mitochondrial content in PeM chicken muscles compared to BPR chicken. This result is inconsistent with more slow muscle fibers bearing a lower mitochondrial content in the PeM. The molecular, cellular and physiological functions of DE genes in the comparison between PeM and BPR include organismal injury, carbohydrate metabolism, cell growth/proliferation, and skeletal muscle system development, indicating that cellular mechanisms in modern broiler lines are tightly associated with rapid growth and differential muscle fiber contents compared to the unselected BPR line. Particularly, PDGF (platelet derived growth factor) signaling and NFE2L2 (nuclear factor, erythroid 2-like 2; also known as NRF2) mediated oxidative stress response pathways appear to be activated in modern broiler compared to the foundational BPR line. Upstream and network analyses revealed that the MSTN (myostatin) –FST (follistatin) interactions and inhibition of AR (androgen receptor) were predicted to be effective regulatory factors for DE genes in modern broiler line. PRKAG3 (protein kinase, AMP-activated, gamma 3 non-catalytic subunit) and LIPE (lipase E) are predicted as core regulatory factors for myogenic development, nutrient and lipid metabolism.ConclusionThe highly upregulated genes in PeM may represent phenotypes of subclinical myopathy commonly observed in the commercial broiler breast tissue, that can lead to muscle hardening, named as woody breast. By investigating global gene expression in a highly selected pedigree broiler line and a foundational breed (Barred Plymouth Rock), the results provide insight into cellular mechanisms that regulate muscle growth, fiber composition and feed efficiency.

Research Article Decrypting the microRNA signatures in gastric cancer using high-throughput miRNA array coupled with systems biological approaches for precision medicine

MicroRNAs (miRNAs) are non-coding small RNA molecules that regulate the differential expression of genes. The expression of miRNAs is dysregulated in various types of cancers, including Gastric Cancer (GC), and has both prognostic and diagnostic potential. To interpret the role of miRNA expression in GC, we evaluated the expression profile using high-throughput miRNA array followed by in-depth systems biological analyses. Total RNA was isolated from 34 GC patients and 15 normal controls and hybridised and differentially expressed miRNAs in GC compared to normal controls were analysed. A total of 250 miRNAs were found to be differentially expressed in GC with a fold change ranging from + 2.5984 to – 3.5271 compared to normal gastric tissues. The differentially expressed miRNAs were further subjected to Ingenuity Pathway Analysis to understand the modulation of cellular and molecular functions and associated physiological system development and functions in GC. The Oncomirs such as miR-155-5p, miR-17-5p, miR-21-5p, miR-23a-3p, miR-24-3p, and miR-320b were significantly (P<0.05) upregulated and all the other miRNAs, mostly tumor suppressors, were significantly downregulated (P<0.05) leading to the reduction in cell death and the induction of gastric cancer, cellular growth and proliferation, cellular movement, cellular assembly and organization, DNA replication, recombination and repair, organismal injury and abnormalities and metastasis of tumor in GC compared to normal tissues. The high-throughput miRNA microarray coupled with systems biological analyses showed that miRNAs are dysregulated in GC, causing the initiation, progression, and metastasis of GC. In conclusion, the novel GC-inducing miRNA signatures decrypted in our study can further be exploited for precision medicine in clinics.

Characterization of the serum and liver proteomes in gut-microbiota-lacking mice.

Current nutrition research is focusing on health promotion, disease prevention, and performance improvement for individuals and communities around the world. The humans with required nutritional ingredients depend on both how well the individual is provided with balanced foods and what state of gut microbiota the host has. Studying the mutually beneficial relationships between gut microbiome and host is an increasing attention in biomedical science. The purpose of this study is to understand the role of gut microbiota and to study interactions between gut microbiota and host. In this study, we used a shotgun proteomic approach to reveal the serum and liver proteomes in gut-microbiota-lacking mice. For serum, 15 and 8 proteins were uniquely detected in specific-pathogen-free (SPF) and germ-free (GF) mice, respectively, as well as the 3 and 20 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Among the proteins of the serum, major urinary protein 1 (MUP-1) of GF mice was significantly decreased compared to SPF mice. In addition, MUP-1 expression is primarily regulated by testosterone. Lacking in gut flora has been implicated in many adverse effects, and now we have found its pathogenic root maybe gut bacteria can regulate the sex-hormone testosterone levels. In the liver, 8 and 22 proteins were uniquely detected in GF mice and SPF mice, respectively, as well as the 14 and 30 proteins were significantly increased and decreased, respectively, in GF mice compared to SPF mice. Furthermore, ingenuity pathway analysis (IPA) indicated that gut microbiota influence the host in cancer, organismal injury and abnormalities, respiratory disease; cell cycle, cellular movement and tissue development; cardiovascular disease, reproductive system disease; and lipid metabolism, molecular transport and small molecule biochemistry. Our findings provide more detailed information of the role of gut microbiota and will be useful to help study gut bacteria and disease prevention.

P3.01-014 Differential Gene Expression of Lung Adenocarcinoma Histology Subtypes According to the IASLC/ATS/ERS Classification: Topic: Morphology

The current lung cancer classification from the IASLC/ATS/ERS integrates lung invasive adenocarcinoma subtypes accounting for the clinical, radiological, molecular and prognostic differences with its implications within the clinical practice. We analyzed the differences in genetic expression of the adenocarcinoma subtypes according to the new WHO 2015 classification. A cohort of 29 NSCLC patients treated at the Instituto Nacional de Cancerología of Mexico from 2008 to 2011. All patients had an available biopsy sample and were classified in four different subtypes of adenocarcinoma (2015 WHO classification). All the tissue samples were analyzed by microarrays to characterize the different expressed genes. IPA Software was used to identify biological processes, functions and biomarkers. Lepidic predominant adenocarcinoma subtype was the only pattern that showed a marked gene expression difference against all predominant histologic patterns, revealing genes with significant (p < 0.01) expression. For all the histological predominant pattern subtype comparisons the top functional networks were related to eight different biological categories as follows: DNA replication; Recombination and Repair; Cell Cycle; Cell Death and Survival; RNA Post-Transcriptional Modification; Cancer; Organismal Injury and Abnormalities; Cellular Development. Moreover, we observed 13 genes with specific differential expression in the Lepidic predominant adenocarcinoma subtype. Lepidic predominant histological pattern subtype has a differential gene expression profile when compared against all predominant histological patterns subtypes. Moreover, we found a gene expression signature of 13 genes that has a unique behavior in the Lepidic histologic pattern subtype that could be used as a specific gene expression signature, biomarker or therapeutic target.