The IBD-associated long noncoding RNA IFNG-AS1 regulates the balance between inflammatory and anti-inflammatory cytokine production after T-cell stimulation.

The inflammatory bowel diseases (IBD) are a complex set of chronic gastrointestinal inflammatory conditions arising from the interplay of genetic and environmental factors. This study focuses on noncoding RNA transcripts as potential mediators of IBD pathophysiology. One particular gene, interferon γ-antisense 1 (IFNG-AS1), has been consistently observed to be elevated in the intestinal mucosa of patients with actively inflamed IBD versus healthy controls. This study builds on these observations, demonstrating that the second splice variant is specifically altered, and this alteration even stratifies within inflamed patients. With the use of a CRISPR-based overexpression system, IFNG-AS1 was selectively overexpressed directly from its genomic loci in T cells. An unbiased mRNA array on these cells identified a large increase in many inflammatory cytokines and a decrease in anti-inflammatory cytokines after IFNG-AS1 overexpression. Media from T cells overexpressing IFNG-AS1 elicited an inflammatory signaling cascade in primary human peripheral blood mononuclear cells, suggesting the potential functional importance of IFNG-AS1 in IBD pathophysiology. The significance of these results is amplified by studies suggesting that a single-nucleotide polymorphism in IFNG-AS1, rs7134599, was associated with both subtypes of patients with IBD independently of race.NEW & NOTEWORTHY Long noncoding RNAs are an emerging field of inflammatory bowel disease (IBD) research. This study mechanistically analyzes the role of a commonly upregulated gene in IBD and shows IFNG-AS1 as a mediator of an inflammatory signaling cascade.

Role of G protein-coupled receptors-microRNA interactions in gastrointestinal pathophysiology.

G protein-coupled receptors (GPCRs) make up the largest transmembrane receptor superfamily in the human genome and are expressed in nearly all gastrointestinal cell types. Coupling of GPCRs and their respective ligands activates various phosphotransferases in the cytoplasm, and, thus, activation of GPCR signaling in intestine regulates many cellular and physiological processes. Studies in microRNAs (miRNAs) demonstrate that they represent critical epigenetic regulators of different pathophysiological responses in different organs and cell types in humans and animals. Here, we reviewed recent research on GPCR-miRNA interactions related to gastrointestinal pathophysiology, such as inflammatory bowel diseases, irritable bowel syndrome, and gastrointestinal cancers. Given that the presence of different types of cells in the gastrointestinal tract suggests the importance of cell-cell interactions in maintaining gastrointestinal homeostasis, we also discuss how GPCR-miRNA interactions regulate gene expression at the cellular level and subsequently modulate gastrointestinal pathophysiology through molecular regulatory circuits and cell-cell interactions. These studies helped identify novel molecular pathways leading to the discovery of potential biomarkers for gastrointestinal diseases.

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR.

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.