Therapeutic potential of miRNAs in Clostridioides difficile infection

Treating Clostridioides difficile infection with miRNAs alone or combined with live biotherapeutic products may augment therapeutic efficacy and help counteract drug resistance in the future

Lin28A and Lin28B Inhibit let-7 MicroRNA Biogenesis by Distinct Mechanisms

SummaryLin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUT4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a Zcchc11-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts are restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors, whereas Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function and have implications for the development of new strategies for cancer therapy

Assessment of Circulating MicroRNAs for the Diagnosis and Disease Activity Evaluation in Patients with Ulcerative Colitis by Using the Nanostring Technology

Background: Clinical decision and patient care management in inflammatory bowel diseases is largely based on the assessment of clinical symptoms, while the biomarkers currently in use poorly reflect the actual disease activity. Therefore, the identification of novel biomarkers will serve an unmet clinical need for IBD screening and patient management. We examined the utility of circulating microRNAs for diagnosis and disease activity monitoring in ulcerative colitis (UC) patients. Methods: Blood serum microRNAs were isolated from UC patients with active and inactive disease and healthy donors. High-throughput microRNA profiling was performed using the Nanostring technology platform. Clinical disease activity was captured by calculating the partial Mayo score. C-reactive protein (CRP) was measured in UC patients as part of their clinical monitoring. The profiles of circulating microRNAs and CRP were correlated with clinical disease indices. Results: We have identified a signature of 12 circulating microRNAs that differentiate UC patients from control subjects. Moreover, six of these microRNAs significantly correlated with UC disease activity. Importantly, a set of four microRNAs (hsa-miR-4454, hsa-miR-223-3p, hsa-miR-23a-3p, and hsa-miR-320e) which correlated with UC disease activity, were found to have higher sensitivity and specificity values than CRP. Conclusions: Circulating microRNAs provide a novel diagnostic and prognostic marker for UC patients. The use of an FDA approved platform could accelerate the application of microRNA screening in a GI clinical setting. When used in combination with current diagnostic and disease activity assessment modalities, microRNAs could improve both IBD screening and care management

Phosphoproteomics Screen Reveals Akt Isoform-Specific Signals Linking RNA Processing to Lung Cancer

The three Akt isoforms are functionally distinct. Here we show that their phosphoproteomes also differ, suggesting that their functional differences are due to differences in target specificity. One of the top cellular functions differentially regulated by Akt isoforms is RNA processing. IWS1, an RNA processing regulator, is phosphorylated by Akt3 and Akt1 at Ser720/Thr721. The latter is required for the recruitment of SETD2 to the RNA Pol II complex. SETD2 trimethylates histone H3 at K36 during transcription, creating a docking site for MRG15 and PTB. H3K36me3-bound MRG15 and PTB regulate FGFR-2 splicing, which controls tumor growth and invasiveness downstream of IWS1 phosphorylation. Twenty-one of the twenty-four non-small-cell-lung carcinomas we analyzed express IWS1. More importantly, the stoichiometry of IWS1 phosphorylation in these tumors correlates with the FGFR-2 splicing pattern and with Akt phosphorylation and Akt3 expression. These data identify an Akt isoform-dependent regulatory mechanism for RNA processing and demonstrate its role in lung cancer

A long noncoding RNA signature for ulcerative colitis identifies IFNG-AS1 as an enhancer of inflammation

High-throughput technologies revealed new categories of genes, including the long noncoding RNAs (lncRNAs), involved in the pathogenesis of human disease; however, the role of lncRNAs in the ulcerative colitis (UC) has not been evaluated. Gene expression profiling was used to develop lncRNA signatures in UC samples. Jurkat T cells were activated by PMA/ionomycin subsequently interferon- (IFNG) and tumor necrosis factor (TNF)- protein levels were assessed by ELISA. Anti-sense molecules were designed to block IFNG-AS1 expression. A unique set of lncRNAs was differentially expressed between UC and control samples. Of these, IFNG-AS1 was among the highest statistically significant lncRNAs (fold change: 5.27, P value: 7.07E-06). Bioinformatic analysis showed that IFNG-AS1 was associated with the IBD susceptibility loci SNP rs7134599 and its genomic location is adjacent to the inflammatory cytokine IFNG. In mouse models of colitis, active colitis samples had increased colonic expression of this lncRNA. Utilizing the Jurkat T cell model, we found IFNG-AS1 to positively regulate IFNG expression. Novel lncRNA signatures differentiate UC patients with active disease, patients in remission, and control subjects. A subset of these lncRNAs was found to be associated with the clinically validated IBD susceptibility loci. IFNG-AS1 was one of these differentially expressed lncRNAs in UC patients and found to regulate the key inflammatory cytokine, IFNG, in CD4 T cells. Taking these findings together, our study revealed novel lncRNA signatures deregulated in UC and identified IFNG-AS1 as a novel regulator of IFNG inflammatory responses, suggesting the potential importance of noncoding RNA mechanisms on regulation of inflammatory bowel disease-related inflammatory responses

MicroRNA-124 Regulates STAT3 Expression and Is Down-regulated in Colon Tissues of Pediatric Patients With Ulcerative Colitis

Background & Aims - Altered levels and functions of microRNAs (miRs) have been associated with inflammatory bowel diseases (IBDs), although little is known about their roles in pediatric IBD. We investigated whether colonic mucosal miRs are altered in children with ulcerative colitis (UC). Methods - We used a library of 316 miRs to identify those that regulate phosphorylation of STAT3 in NCM460 human colonocytes incubated with interleukin-6. Levels of miR-124 were measured by real-time PCR analysis of colon biopsies from pediatric and adult patients with UC and patients without IBD (controls), and of HCT-116 colonocytes incubated with 5-aza-2’-deoxycytidine. Methylation of the MIR124 promoter was measured by quantitative methylation-specific PCR. Results - Levels of phosphorylated STAT3 and the genes it regulates (encoding VEGF, BCL2, BCLXL, and MMP9) were increased in pediatric patients with UC, compared to control tissues. Overexpression of miR-124, let-7, miR-125, miR-26, or miR-101 reduced STAT3 phosphorylation by ≥75% in NCM460 cells; miR-124 had the greatest effect. miR-124 was downregulated specifically in colon tissues from pediatric patients with UC and directly targeted STAT3 mRNA. Levels of miR-124 were decreased whereas levels of STAT3 phosphorylation increased in colon tissues from pediatric patients with active UC, compared to those with inactive disease. Furthermore, levels of miR-124 and STAT3 were inversely correlated in mice with experimental colitis. Downregulation of miR-124 in tissues from children with UC was attributed to hypermethylation of its promoter region. Incubation of HCT-116 colonocytes with 5-aza-2’ deoxycytidine upregulated miR-124 and reduced levels of STAT3 mRNA. Conclusions - MiR-124 appears to regulate the expression of STAT3. Reduced levels of miR-124 in colon tissues of children with active UC appear to increase expression and activity of STAT3, which could promote inflammation and pathogenesis of UC in children

miR-24 is elevated in ulcerative colitis patients and regulates intestinal epithelial barrier function

Inflammatory bowel disease is characterized by high levels of inflammation and loss of barrier integrity in the colon. The intestinal barrier is a dynamic network of proteins that encircle intestinal epithelial cells. miRNAs regulate protein-coding genes. In this study, miR-24 was found to be elevated in colonic biopsies and blood samples from ulcerative colitis (UC) patients compared with healthy controls. In the colon of UC patients, miR-24 is localized to intestinal epithelial cells, which prompted an investigation of intestinal epithelial barrier function. Two intestinal epithelial cell lines were used to study the effect of miR-24 overexpression on barrier integrity. Overexpression of miR-24 in both cell lines led to diminished transepithelial electrical resistance and increased dextran flux, suggesting an effect on barrier integrity. Overexpression of miR-24 did not induce apoptosis or affect cell proliferation, suggesting that the effect of miR-24 on barrier function was due to an effect on cell–cell junctions. Although the tight junctions in cells overexpressing miR-24 appeared normal, miR-24 overexpression led to a decrease in the tight junction–associated protein cingulin. Loss of cingulin compromised barrier formation; cingulin levels negatively correlated with disease severity in UC patients. Together, these data suggest that miR-24 is a significant regulator of intestinal barrier that may be important in the pathogenesis of UC

The effect of sex and irritable bowel syndrome on HPA axis response and peripheral glucocorticoid receptor expression

Background & Aims: Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis has been reported in irritable bowel syndrome (IBS). Enhanced HPA axis responses have been associated with reduced glucocorticoid receptor (GR) mediated negative feedback inhibition. We aimed to study the effects of IBS status, sex, or presence of early adverse life events (EAL) on the cortisol response to corticotropin-releasing factor (CRF) and adrenocorticotropic hormone (ACTH), and on GR mRNA expression in peripheral blood mononuclear cells (PBMCs). Methods: Rome III+ IBS patients and healthy controls underwent CRF (1μg/kg ovine) and ACTH (250μg) stimulation tests with serial plasma ACTH and cortisol levels measured (n=116). GR mRNA levels were measured using quantitative PCR (n=143). Area under the curve (AUC) and linear mixed effects models were used to compare ACTH and cortisol response measured across time between groups. Results: There were divergent effects of IBS on the cortisol response to ACTH by sex. In men, IBS was associated with an increased AUC (p= 0.009), but in women AUC was blunted in IBS(p=0.006). Men also had reduced GR mRNA expression (p=0.007). Cumulative exposure to EALs was associated with an increased HPA response. Lower GR mRNA was associated with increased pituitary HPA response and increased severity of overall symptoms and abdominal pain in IBS. Conclusion: This study highlights the importance of considering sex in studies of IBS and the stress response in general. Our findings also provide support for PBMC GR mRNA expression as a peripheral marker of central HPA response

A Multi-Factorial Observational Study on Sequential Fecal Microbiota Transplant in Patients with Medically Refractory Clostridioides difficile Infection

Fecal microbiota transplantation (FMT) is highly effective in recurrent Clostridioides difficile infection (CDI); increasing evidence supports FMT in severe or fulminant Clostridioides difficile infection (SFCDI). However, the multifactorial mechanisms that underpin the efficacy of FMT are not fully understood. Systems biology approaches using high-throughput technologies may help with mechanistic dissection of host-microbial interactions. Here, we have undertaken a deep phenomics study on four adults receiving sequential FMT for SFCDI, in which we performed a longitudinal, integrative analysis of multiple host factors and intestinal microbiome changes. Stool samples were profiled for changes in gut microbiota and metabolites and blood samples for alterations in targeted epigenomic, metabonomic, glycomic, immune proteomic, immunophenotyping, immune functional assays, and T-cell receptor (TCR) repertoires, respectively. We characterised temporal trajectories in gut microbial and host immunometabolic data sets in three responders and one non-responder to sequential FMT. A total of 562 features were used for analysis, of which 78 features were identified, which differed between the responders and the non-responder. The observed dynamic phenotypic changes may potentially suggest immunosenescent signals in the non-responder and may help to underpin the mechanisms accompanying successful FMT, although our study is limited by a small sample size and significant heterogeneity in patient baseline characteristics. Our multi-omics integrative longitudinal analytical approach extends the knowledge regarding mechanisms of efficacy of FMT and highlights preliminary novel signatures, which should be validated in larger studies