Interferon-γ signaling in eosinophilic esophagitis has implications for epithelial barrier function and programmed cell death.

Objective: Eosinophilic esophagitis (EoE) is a chronic esophageal inflammatory disorder characterized by eosinophil-rich mucosal inflammation and tissue remodeling. Transcriptional profiling of esophageal biopsies has previously revealed upregulation of type I and II interferon (IFN) response genes. We aim to unravel interactions between immune and epithelial cells and examine functional significance in esophageal epithelial cells. Design: We investigated epithelial gene expression from EoE patients using single-cell RNA sequencing and a confirmatory bulk RNA-sequencing experiment of isolated epithelial cells. The functional impact of interferon signaling on epithelial cells was investigated using in vitro organoid models. Results: We observe upregulation of interferon response signature genes (ISGs) in the esophageal epithelium during active EoE compared to other cell types, single-cell data, and pathway analyses, identified upregulation in ISGs in epithelial cells isolated from EoE patients. Using an esophageal organoid and air-liquid interface models, we demonstrate that IFN-γ stimulation triggered disruption of esophageal epithelial differentiation, barrier integrity, and induced apoptosis via caspase upregulation. We show that an increase in cleaved caspase-3 is seen in EoE tissue and identify interferon gamma (IFNG) expression predominantly in a cluster of majority-CD8+ T cells with high expression of CD69 and FOS. Conclusion: These findings offer insight into the interplay between immune and epithelial cells in EoE. Our data illustrate the relevance of several IFN-γ-mediated mechanisms on epithelial function in the esophagus, which have the potential to impact epithelial function during inflammatory conditions.

Persistent esophageal changes after histologic remission in eosinophilic esophagitis.

BACKGROUND: Eosinophilic esophagitis (EoE) is characterized by persistent or relapsing allergic inflammation, and both clinical and histologic features of esophageal inflammation persist over time in most individuals. Mechanisms contributing to EoE relapse are not understood, and chronic EoE-directed therapy is therefore required to prevent long-term sequelae. OBJECTIVE: We investigated whether EoE patients in histologic remission have persistent dysregulation of esophageal gene expression. METHODS: Esophageal biopsy samples from 51 pediatric and 52 adult subjects with EoE in histopathologic remission (<15 eosinophils per high-power field [eos/hpf]) and control (48 pediatric and 167 adult) subjects from multiple institutions were subjected to molecular profiling by the EoE diagnostic panel, which comprises a set of 94 esophageal transcripts differentially expressed in active EoE. RESULTS: Defining remission as <15 eos/hpf, we identified 51 and 32 differentially expressed genes in pediatric and adult EoE patients compared to control individuals, respectively (false discovery rate < 0.05). Using the stringent definition of remission (0 eos/hpf), the adult and pediatric cohorts continued to have 18 and 25 differentially expressed genes (false discovery rate < 0.05). Among 6 shared genes between adults and children, CDH26 was upregulated in both children and adults; immunohistochemistry demonstrated increased cadherin 26 staining in the epithelium of EoE patients in remission compared to non-EoE controls. In the adult cohort, POSTN expression correlated with the endoscopic reference system score (Spearman r = 0.35, P = .011), specifically correlating with the rings' endoscopic reference system subscore (r = 0.53, P = .004). CONCLUSION: We have identified persistent EoE-associated esophageal gene expression in patients with disease in deep remission. These data suggest potential inflammation-induced epigenetic mechanisms may influence gene expression during remission in EoE and provide insight into possible mechanisms that underlie relapse in EoE.

Defective quality control autophagy in Hyperhomocysteinemia promotes ER stress and consequent neuronal apoptosis through proteotoxicity.

Homocysteine (Hcy), produced physiologically in all cells, is an intermediate metabolite of methionine and cysteine metabolism. Hyperhomocysteinemia (HHcy) resulting from an in-born error of metabolism that leads to accumulation of high levels of Hcy, is associated with vascular damage, neurodegeneration and cognitive decline. Using a HHcy model in neuronal cells, primary cortical neurons and transgenic zebrafish, we demonstrate diminished autophagy and Hcy-induced neurotoxicity associated with mitochondrial dysfunction, fragmentation and apoptosis. We find this mitochondrial dysfunction is due to Hcy-induced proteotoxicity leading to ER stress. We show this sustained proteotoxicity originates from the perturbation of upstream autophagic pathways through an aberrant activation of mTOR and that protetoxic stress act as a feedforward cues to aggravate a sustained ER stress that culminate to mitochondrial apoptosis in HHcy model systems. Using chemical chaperones to mitigate sustained ER stress, Hcy-induced proteotoxicity and consequent neurotoxicity were rescued. We also rescue neuronal lethality by activation of autophagy and thereby reducing proteotoxicity and ER stress. Our findings pave the way to devise new strategies for the treatment of neural and cognitive pathologies reported in HHcy, by either activation of upstream autophagy or by suppression of downstream ER stress. Video Abstract.

Diagnostic and Prognostic Potential of MiR-379/656 MicroRNA Cluster in Molecular Subtypes of Breast Cancer.

INTRODUCTION: Breast cancer is the most frequently diagnosed cancer globally and is one of the most important contributors to cancer-related deaths. Earlier diagnosis is known to reduce mortality, and better biomarkers are needed. MiRNA clusters often co-express and target mRNAs in a coordinated fashion, perturbing entire pathways; they thus merit further exploration for diagnostic or prognostic use. MiR-379/656, at chromosome 14q32, is the second largest miRNA cluster in the human genome and implicated in various malignancies including glioblastoma, melanoma, gastrointestinal tumors and ovarian cancer highlighting its potential importance. In this study, we focus on the diagnostic and prognostic potentials of MiR-379/656 in breast cancer and its molecular subtypes. MATERIALS AND METHODS: We analyzed miRNA and mRNA next generation sequencing data from 903 primary tumors and 90 normal controls (source: The Cancer Genome Atlas). The differential expression profile between tumor and normal was analyzed using DeSEQ2. Penalized logistic regression modelling (lasso regression) was used to assess the predictive potential of MiR-379/656 expression for tumor and normal samples. The association between MiR-379/656 expression and overall patient survival was studied using Cox Proportional-Hazard Model. The target mRNAs (validated) of MiR-379/656 were annotated via pathway enrichment analysis to understand the biological significance of the cluster in breast cancer. RESULTS: The differential expression analysis for 1390 miRNAs (miRnome) revealed 310 upregulated (22.3%) and 176 downregulated (12.66%) miRNAs in breast cancer patients compared with controls. For MiR-379/656, 32 miRNAs (32/42; 76%) were downregulated. The MiR-379/656 cluster was found to be the most differentially expressed cluster in the human genome (p < 10-30). The Basal and Luminal B subtypes showed at least 83% (35/42) of the miRNAs to be downregulated. The binomial model prioritized 15 miRNAs, which distinguished breast cancer patients from controls with 99.15 ± 0.58% sensitivity and 77.78 ± 5.24% specificity. Overall, the Basal and Luminal B showed the most effective predictive power with respect to the 15 prioritized miRNAs at MiR-379/656 cluster. The decreased expression of MiR-379/656 was found to be associated with poorer clinical outcome in Basal and Luminal B subtypes, increasing tumor stage and tumor size/extent, and overall patient survival. Pathway enrichment for the validated targets of MiR-379/656 was significant for cancer-related pathways, especially DNA repair, transcriptional regulation by p53 and cell cycle checkpoints (adjusted p-value < 0.05). CONCLUSIONS: Genome informatics analysis of high throughput data for MiR-379/656 cluster has shown that a subset of 15 miRNAs from MiR-379/656 cluster can be used for the diagnostic and prognostic purpose of breast cancer and its subtypes-especially in Basal and Luminal B.

Mitochondrial metabolism and calcium homeostasis in the development of NAFLD leading to hepatocellular carcinoma.

Non-alcoholic fatty liver disease (NAFLD) is a metabolic syndrome characterized by excessive accumulation of hepatic lipid droplets. The disease progresses with steatosis as the premise for hepatocytic damage and tissue scarring, often culminating in hepatocellular carcinoma (HCC). Perturbations in mitochondrial metabolism and energetics were found to be associated with, and often instrumental in various stages of this progression. Functional impairment of the mitochondria affects all aspects of cellular functioning and a particularly important one is calcium signalling. Changes in mitochondrial calcium specifically in hepatocytes of a fatty liver, is reflected by alterations in calcium signalling as well as calcium transporter activities. This deranged Ca2+ homeostasis aids in even more uptake of lipids into the mitochondria and a shift in equilibrium, both metabolically as well as in terms of energy production, leading to completely altered cellular states. These alterations have been reviewed as a perspective to understand the disease progression through NAFLD leading to HCC.

Human Brain Shows Recurrent Non-Canonical MicroRNA Editing Events Enriched for Seed Sequence with Possible Functional Consequence.

RNA editing is a post-transcriptional modification, which can provide tissue-specific functions not encoded in DNA. Adenosine-to-inosine is the predominant editing event and, along with cytosine-to-uracil changes, constitutes canonical editing. The rest is non-canonical editing. In this study, we have analysed non-canonical editing of microRNAs in the human brain. We have performed massively parallel small RNA sequencing of frontal cortex (FC) and corpus callosum (CC) pairs from nine normal individuals (post-mortem). We found 113 and 90 unique non-canonical editing events in FC and CC samples, respectively. More than 70% of events were in the miRNA seed sequence-implicating an altered set of target mRNAs and possibly resulting in a functional consequence. Up to 15% of these events were recurring and found in at least three samples, also supporting the biological relevance of such variations. Two specific sequence variations, C-to-A and G-to-U, accounted for over 80% of non-canonical miRNA editing events-and revealed preferred sequence motifs. Our study is one of the first reporting non-canonical editing in miRNAs in the human brain. Our results implicate miRNA non-canonical editing as one of the contributing factors towards transcriptomic diversity in the human brain.

Crosstalk Between the Unfolded Protein Response, MicroRNAs, and Insulin Signaling Pathways: In Search of Biomarkers for the Diagnosis and Treatment of Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder that is characterized by functional defects in glucose metabolism and insulin secretion. Its complex etiology and multifaceted nature have made it difficult to design effective therapies for early diagnosis and treatment. Several lines of evidence indicate that aberrant activation of the unfolded protein response (UPR) in response to endoplasmic reticulum (ER) stress impairs the ß cell's ability to respond to glucose and promotes apoptosis. Elucidating the molecular mechanisms that govern ß cell dysfunction and cell death can help investigators design therapies to halt or prevent the development of T2DM. Early diagnosis of T2DM, however, warrants additionally the identification of potential biomarkers. MicroRNAs (miRNAs) are key regulators of transcriptional processes that modulate various features of insulin signaling, such as insulin sensitivity, glucose tolerance, and insulin secretion. A deeper understanding of how changes in patterns of expression of miRNAs correlate with altered glucose metabolism can enable investigators to develop methods for the early diagnosis and treatment of T2DM. The first part of this review examines how altered expression of specific UPR pathway proteins disrupts ER function and causes ß cell dysfunction, while the second part discusses the potential role of miRNAs in the diagnostic and treatment of T2DM.

A-to-I editing in human miRNAs is enriched in seed sequence, influenced by sequence contexts and significantly hypoedited in glioblastoma multiforme.

Editing in microRNAs, particularly in seed can significantly alter the choice of their target genes. We show that out of 13 different human tissues, different regions of brain showed higher adenosine to inosine (A-to-I) editing in mature miRNAs. These events were enriched in seed sequence (73.33%), which was not observed for cytosine to uracil (17.86%) editing. More than half of the edited miRNAs showed increased stability, 72.7% of which had ΔΔG values less than -6.0 Kcal/mole and for all of them the edited adenosines mis-paired with cytosines on the pre-miRNA structure. A seed-editing event in hsa-miR-411 (with A - C mismatch) lead to increased expression of the mature form compared to the unedited version in cell culture experiments. Further, small RNA sequencing of GBM patients identified significant miRNA hypoediting which correlated with downregulation of ADAR2 both in metadata and qRT-PCR based validation. Twenty-two significant (11 novel) A-to-I hypoediting events were identified in GBM samples. This study highlights the importance of specific sequence and structural requirements of pre-miRNA for editing along with a suggestive crucial role for ADAR2. Enrichment of A-to-I editing in seed sequence highlights this as an important layer for genomic regulation in health and disease, especially in human brain.