NKX2-3 transcriptional regulation of endothelin-1 and VEGF signaling in human intestinal microvascular endothelial cells

BACKGROUND: NKX2-3 is associated with inflammatory bowel disease (IBD). NKX2-3 is expressed in microvascular endothelial cells and the muscularis mucosa of the gastrointestinal tract. Human intestinal microvascular endothelial cells (HIMECs) are actively involved in the pathogenesis of IBD and IBD-associated microvascular dysfunction. To understand the cellular function of NKX2-3 and its potential role underlying IBD pathogenesis, we investigated the genes regulated by NKX2-3 in HIMEC using cDNA microarray. METHODOLOGY/PRINCIPAL FINDINGS: NKX2-3 expression was suppressed by shRNA in two HIMEC lines and gene expression was profiled by cDNA microarray. Pathway Analysis was used to identify gene networks according to biological functions and associated pathways. Validation of microarray and genes expression in intestinal tissues was assessed by RT-PCR. NKX2-3 regulated genes are involved in immune and inflammatory response, cell proliferation and growth, metabolic process, and angiogenesis. Several inflammation and angiogenesis related signaling pathways that play important roles in IBD were regulated by NKX2-3, including endothelin-1 and VEGF-PI3K/AKT-eNOS. Expression levels of NKX2-3, VEGFA, PI3K, AKT, and eNOS are increased in intestinal tissues from IBD patients and expression levels of EDN1 are decreased in intestinal tissues from IBD patients. These results demonstrated the important roles of NKX2-3, VEGF, PI3K, AKT, eNOS, and EDN1 in IBD pathogenesis. Correlation analysis showed a positive correlation between mRNA expression of NKX2-3 and VEGFA and a negative correlation between mRNA expression of NKX2-3 and EDN1 in intestinal tissues from IBD patients. CONCLUSION/RELEVANCE: NKX2-3 may play an important role in IBD pathogenesis by regulating endothelin-1 and VEGF signaling in HIMECs.     

In vivo effects on intron retention and exon skipping by the U2AF large subunit and SF1/BBP in the nematode Caenorhabditis elegans

The in vivo analysis of the roles of splicing factors in regulating alternative splicing in animals remains a challenge. Using a microarray-based screen, we identified a Caenorhabditis elegans gene, tos-1, that exhibited three of the four major types of alternative splicing: intron retention, exon skipping, and, in the presence of U2AF large subunit mutations, the use of alternative 3' splice sites. Mutations in the splicing factors U2AF large subunit and SF1/BBP altered the splicing of tos-1. 3' splice sites of the retained intron or before the skipped exon regulate the splicing pattern of tos-1. Our study provides in vivo evidence that intron retention and exon skipping can be regulated largely by the identities of 3' splice sites.     

Genotypes and haplotypes in the insulin-like growth factors, their receptors and binding proteins in relation to plasma metabolic levels and mammographic density

Background

Chronic inflammatory diseases including inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), psoriasis and rheumatoid arthritis (RA) afflict millions of people worldwide, but their pathogenesis is still not well understood. It is also not well known if distinct changes in gene expression characterize these diseases and if these patterns can discriminate between diseased and control patients and/or stratify the disease. The main focus of our work was the identification of novel markers that overlap among the 3 diseases or discriminate them from each other.

Methods

Diseased (n = 13, n = 15 and n = 12 in IBD, psoriasis and RA respectively) and healthy patients (n = 18) were recruited based on strict inclusion and exclusion criteria; peripheral blood samples were collected by clinicians (30 ml) in Venous Blood Vacuum Collection Tubes containing EDTA and peripheral blood mononuclear cells were separated by Ficoll gradient centrifugation. RNA was extracted using Trizol reagent. Gene expression data was obtained using TaqMan Low Density Array (TLDA) containing 96 genes that were selected by an algorithm and the statistical analyses were performed in Prism by using non-parametric Mann-Whitney U test (P-values < 0.05).

Results

Here we show that using a panel of 96 disease associated genes and measuring mRNA expression levels in peripheral blood derived mononuclear cells; we could identify disease-specific gene panels that separate each disease from healthy controls. In addition, a panel of five genes such as ADM, AQP9, CXCL2, IL10 and NAMPT discriminates between all samples from patients with chronic inflammation and healthy controls. We also found genes that stratify the diseases and separate different subtypes or different states of prognosis in each condition.

Conclusions

These findings and the identification of five universal markers of chronic inflammation suggest that these diseases have a common background in pathomechanism, but still can be separated by peripheral blood gene expression. Importantly, the identified genes can be associated with overlapping biological processes including changed inflammatory response. Gene panels based on such markers can play a major role in the development of personalized medicine, in monitoring disease progression and can lead to the identification of new potential drug targets in chronic inflammation.     

SF3B4 promotes ovarian cancer progression by regulating alternative splicing of RAD52

Many studies have proven that splicing factors are crucial for human malignant tumor development. However, as a classical splicing factor, the expression of SF3B4 is not clear, and its biological function needs to be further clarified in ovarian cancer (OC). We determined that SF3B4 was obviously upregulated and its high expression was associated with poor prognosis in OC patients. In vitro and in vivo assays suggested that SF3B4 overexpression promoted OC cell proliferation and mobility, and downregulation of SF3B4 had the opposite effect. Further studies found that miR-509–3p decreased SF3B4 mRNA expression by binding to the 3’ -UTR of SF3B4 directly. Importantly, we revealed that RAD52 was a potential target of SF3B4 through alternative splicing events analysis. Loss of SF3B4 led to decreased expression of RAD52, owing to intron 8 retention and generation of premature termination codons. Moreover, decreased expression of RAD52 partially counteracted the tumor-promoting effect of SF3B4 overexpression. In conclusion, our results suggested that SF3B4, negatively regulated by miR-509–3p, promoted OC progression through effective splicing of RAD52. Therefore, SF3B4 may be a promising biomarker and effective therapeutic target for OC.Subject terms: Ovarian cancer, Prognostic markers     

Pathogenic angiogenesis in IBD and experimental colitis: new ideas and therapeutic avenues

Angiogenesis is now understood to play a major role in the pathology of chronic inflammatory diseases and is indicated to exacerbate disease pathology. Recent evidence shows that angiogenesis is crucial during inflammatory bowel disease (IBD) and in experimental models of colitis. Examination of the relationship between angiogenesis and inflammation in experimental colitis shows that initiating factors for these responses simultaneously increase as disease progresses and correlate in magnitude. Recent studies show that inhibition of the inflammatory response attenuates angiogenesis to a similar degree and, importantly, that inhibition of angiogenesis does the same to inflammation. Recent data provide evidence that differential regulation of the angiogenic mediators involved in IBD-associated chronic inflammation is the root of this pathological angiogenesis. Many factors are involved in this phenomenon, including growth factors/cytokines, chemokines, adhesion molecules, integrins, matrix-associated molecules, and signaling targets. These factors are produced by various vascular, inflammatory, and immune cell types that are involved in IBD pathology. Moreover, recent studies provide evidence that antiangiogenic therapy is a novel and effective approach for IBD treatment. Here we review the role of pathological angiogenesis during IBD and experimental colitis and discuss the therapeutic avenues this recent knowledge has revealed.     

The role of substance P in inflammatory disease

The diffuse neuroendocrine system consists of specialised endocrine cells and peptidergic nerves and is present in all organs of the body. Substance P (SP) is secreted by nerves and inflammatory cells such as macrophages, eosinophils, lymphocytes, and dendritic cells and acts by binding to the neurokinin-1 receptor (NK-1R). SP has proinflammatory effects in immune and epithelial cells and participates in inflammatory diseases of the respiratory, gastrointestinal, and musculoskeletal systems. Many substances induce neuropeptide release from sensory nerves in the lung, including allergen, histamine, prostaglandins, and leukotrienes. Patients with asthma are hyperresponsive to SP and NK-1R expression is increased in their bronchi. Neurogenic inflammation also participates in virus-associated respiratory infection, non-productive cough, allergic rhinitis, and sarcoidosis. SP regulates smooth muscle contractility, epithelial ion transport, vascular permeability, and immune function in the gastrointestinal tract. Elevated levels of SP and upregulated NK-1R expression have been reported in the rectum and colon of patients with inflammatory bowel disease (IBD), and correlate with disease activity. Increased levels of SP are found in the synovial fluid and serum of patients with rheumatoid arthritis (RA) and NK-1R mRNA is upregulated in RA synoviocytes. Glucocorticoids may attenuate neurogenic inflammation by decreasing NK-1R expression in epithelial and inflammatory cells and increasing production of neutral endopeptidase (NEP), an enzyme that degrades SP. Preventing the proinflammatory effects of SP using tachykinin receptor antagonists may have therapeutic potential in inflammatory diseases such as asthma, sarcoidosis, chronic bronchitis, IBD, and RA. In this paper, we review the role that SP plays in inflammatory disease.     

RNA结合蛋白与巨噬细胞炎症因子表达

RNA结合蛋白(RNA-binding proteins, RBPs)是转录后基因表达的关键调控因子,参与剪接、出核、翻译和稳定性等RNA代谢调控。RBPs表达或功能异常可导致炎症性疾病、代谢性疾病以及神经系统疾病等多种疾病的发生发展。炎症是机体对外界刺激及损伤的防御性免疫反应。巨噬细胞作为机体重要的免疫细胞,通过快速响应刺激并且释放大量炎症因子,进而调控炎症反应。巨噬细胞中炎症因子的表达受到转录以及转录后水平的调控。其中,RBPs参与大量RNA的转录后调控过程。研究发现,一方面,RBPs直接结合炎症因子mRNA中的顺式作用元件,参与其mRNA稳定性和翻译等过程,例如TTP(tristetraprolin);另一方面,某些RBPs通过参与炎症信号通路中一些关键基因mRNA的稳定性、翻译或选择性剪接调控,进而间接影响炎症因子表达及分泌。例如,剪接因子3A亚基1(splicing factor 3A subunit 1, SF3A1)。本文主要总结RBPs在mRNA稳定性、翻译和选择性剪接不同转录后水平调控巨噬细胞炎症因子表达的作用机制。这些RBPs从不同的层面直接或者间接参与调控炎症因子...     

Association pattern mining of intron retention events in human based on hybrid learning machine

Alternative splicing is a main component of protein diversity, and aberrant splicing is known to be one of the main causes of genetic disorders such as cancer. Many statistical and computational approaches have identified several major factors that determine the splicing event, such as exon/intron length, splice site strength, and density of splicing enhancers or silencers. These factors may be correlated with one another and thus result in a specific type of splicing, but there has not been a systematic approach to extracting comprehensible association patterns. Here, we attempted to understand the decision making process of the learning machine on intron retention event. We adopted a hybrid learning machine approach using a random forest and association rule mining algorithm to determine the governing factors of intron retention events and their combined effect on decision-making processes. By quantifying all candidate features into five category values, we enhanced the understandability of generated rules. The interesting features found by the random forest algorithm are that only the adenine- and thymine-based triplets such as ATA, TTA, and ATT, but not the known intronic splicing enhancer GGG triplet is shown the significant features. The rules generated by the association rule mining algorithm also show that constitutive introns are generally characterized by high adenine- and thymine-based triplet frequency (level 3 and above), 3' and 5' splice site scores, exonic splicing silencer scores, and intron length, whereas retained introns are characterized by low-level counterpart scores.     

Whole Genome Gene Expression Meta-Analysis of Inflammatory Bowel Disease Colon Mucosa Demonstrates Lack of Major Differences between Crohn's Disease and Ulcerative Colitis

Background

In inflammatory bowel disease (IBD), genetic susceptibility together with environmental factors disturbs gut homeostasis producing chronic inflammation. The two main IBD subtypes are Ulcerative colitis (UC) and Crohn’s disease (CD). We present the to-date largest microarray gene expression study on IBD encompassing both inflamed and un-inflamed colonic tissue. A meta-analysis including all available, comparable data was used to explore important aspects of IBD inflammation, thereby validating consistent gene expression patterns.

Methods

Colon pinch biopsies from IBD patients were analysed using Illumina whole genome gene expression technology. Differential expression (DE) was identified using LIMMA linear model in the R statistical computing environment. Results were enriched for gene ontology (GO) categories. Sets of genes encoding antimicrobial proteins (AMP) and proteins involved in T helper (Th) cell differentiation were used in the interpretation of the results. All available data sets were analysed using the same methods, and results were compared on a global and focused level as t-scores.

Results

Gene expression in inflamed mucosa from UC and CD are remarkably similar. The meta-analysis confirmed this. The patterns of AMP and Th cell-related gene expression were also very similar, except for IL23A which was consistently higher expressed in UC than in CD. Un-inflamed tissue from patients demonstrated minimal differences from healthy controls.

Conclusions

There is no difference in the Th subgroup involvement between UC and CD. Th1/Th17 related expression, with little Th2 differentiation, dominated both diseases. The different IL23A expression between UC and CD suggests an IBD subtype specific role. AMPs, previously little studied, are strongly overexpressed in IBD. The presented meta-analysis provides a sound background for further research on IBD pathobiology.     

Increased expression and activation of IL-12-induced Stat4 signaling in the mucosa of ulcerative colitis patients

Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic inflammatory diseases with unsolved pathogenesis. Imbalanced Th1/Th2 may play a role in the sustained inflammation of IBD. In China, CD is rare but the incidence of UC has been rising steadily in the last two decades. We investigated the expression of IL-12 (p40) and IFN-γ, and the activational state of Stat4 signaling in mucosal tissues at the site of disease from 30 active UC patients in comparison with 30 healthy controls. RT-PCR analyses revealed increased mRNA expression of IL-12 (p40) but not IFN-γ in UC patients. Western blot analyses discovered, for the first time, increased levels of constitutive Stat4 in the cytoplasm and phosphorylated Stat4 in the nucleus of mucosal cells from UC patients. We conclude that a heightened, perhaps persistent, activational state of IL-12/Stat4, and/or IL-23/Stat4 signaling may be present in active Chinese UC patients, and possibly involved in chronic inflammation in UC.     

The Drosophila splicing regulator sex-lethal directly inhibits translation of male-specific-lethal 2 mRNA.

Male-specific expression of the protein male-specific-lethal 2 (MSL-2) controls dosage compensation in Drosophila. msl-2 gene expression is inhibited in females by Sex-lethal (SXL), an RNA binding protein known to regulate pre-mRNA splicing. An intron present at the 5' untranslated region (UTR) of msl-2 mRNA contains putative SXL binding sites and is retained in female flies. Here we show that SXL plays a dual role in the inhibition of msl-2 expression. Cotransfection of Drosophila Schneider cells with an SXL expression vector and a reporter containing the 5' UTR of msl-2 mRNA resulted in retention of the 5' UTR intron and efficient accumulation of the unspliced mRNA in the cytoplasm, where its translation was blocked by SXL, but not by the intron per se. Both splicing and translation inhibition by SXL were recapitulated in vitro and found to be dependent upon SXL binding to high-affinity sites within the intron, showing that SXL directly regulates these events. Our data reveal a coordinated mechanism for the regulation of msl-2 expression by the same regulatory factor: SXL enforces intron retention in the nucleus and subsequent translation inhibition in the cytoplasm.     

Multi–omic analysis of signalling factors in inflammatory comorbidities

Background

Inflammation is a core element of many different, systemic and chronic diseases that usually involve an important autoimmune component. The clinical phase of inflammatory diseases is often the culmination of a long series of pathologic events that started years before. The systemic characteristics and related mechanisms could be investigated through the multi–omic comparative analysis of many inflammatory diseases. Therefore, it is important to use molecular data to study the genesis of the diseases. Here we propose a new methodology to study the relationships between inflammatory diseases and signalling molecules whose dysregulation at molecular levels could lead to systemic pathological events observed in inflammatory diseases.

Results

We first perform an exploratory analysis of gene expression data of a number of diseases that involve a strong inflammatory component. The comparison of gene expression between disease and healthy samples reveals the importance of members of gene families coding for signalling factors. Next, we focus on interested signalling gene families and a subset of inflammation related diseases with multi–omic features including both gene expression and DNA methylation. We introduce a phylogenetic–based multi–omic method to study the relationships between multi–omic features of inflammation related diseases by integrating gene expression, DNA methylation through sequence based phylogeny of the signalling gene families. The models of adaptations between gene expression and DNA methylation can be inferred from pre–estimated evolutionary relationship of a gene family. Members of the gene family whose expression or methylation levels significantly deviate from the model are considered as the potential disease associated genes.

Conclusions

Applying the methodology to four gene families (the chemokine receptor family, the TNF receptor family, the TGF– β gene family, the IL–17 gene family) in nine inflammation related diseases, we identify disease associated genes which exhibit significant dysregulation in gene expression or DNA methylation in the inflammation related diseases, which provides clues for functional associations between the diseases.

     

Wilms’ tumor 1-associating protein complex regulates alternative splicing and polyadenylation at potential G-quadruplex-forming splice site sequences

Wilms’ tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various processes of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. Here we demonstrate using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. We also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3′-end processing complex. Taken together, our results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences.     

The role of heme oxygenase and carbon monoxide in inflammatory bowel disease

Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease, is a chronic and recurrent inflammatory disorder of the intestinal tract. Since the precise pathogenesis of IBD remains unclear, it is important to investigate the pathogenesis of IBD and to evaluate new anti-inflammatory strategies. Recent evidence suggests that heme oxygenase-1 (HO-1) plays a critical protective role during the development of intestinal inflammation. In fact, it has been demonstrated that the activation of HO-1 may act as an endogenous defensive mechanism to reduce inflammation and tissue injury in various animal intestinal injury models induced by ischemia-reperfusion, indomethacin, lipopolysaccharide-associated sepsis, trinitrobenzene sulfonic acid or dextran sulfate sodium. In addition, carbon monoxide (CO) derived from HO-1 has been shown to be involved in the regulation of intestinal inflammation. Furthermore, administration of a low concentration of exogenous CO has a protective effect against intestinal inflammation. These data suggest that HO-1 and CO may be novel therapeutic molecules for patients with gastrointestinal inflammatory diseases. In this review, we present what is currently known regarding the role of HO-1 and CO in intestinal inflammation.