MicroRNA-31 and MicroRNA-155 Are Overexpressed in Ulcerative Colitis and Regulate IL-13 Signaling by Targeting Interleukin 13 Receptor α-1

Interleukin-13 (IL-13) is an important Type 2 T helper (Th2) cytokine, controlling biological functions in epithelium and has been linked to asthma, atopic dermatitis and ulcerative colitis (UC). Interleukin-13 signals through IL-13 receptor α-1 (IL13RA1 (gene) and IL13Rα1 (protein)), a receptor that can be regulated by microRNAs (miRs). MicroRNAs are small non-coding single-stranded RNAs with a role in several pathologies. However, their relevance in the pathophysiology of UC, a chronic inflammatory condition of the colonic mucosa, is poorly characterised. Here, we determined the expression of IL13Rα1 in UC, its potential regulation by miRs and the subsequent effect on IL-13 signalling. Inflamed mucosa of UC patients showed decreased mRNA and protein expression of IL13RA1 when compared to healthy controls. We show that miR-31 and miR-155 are upregulated in inflamed UC mucosa and that both directly target the 3′ untranslated region of IL13RA1 mRNA. Transfection of miR-31 and miR-155 mimics reduced the expression of IL13RA1 mRNA and protein, and blocked IL-13-dependent phosphorylation of signal transducer and activator of transcription 6 (STAT6) in HT-29 cells, a gut epithelium cell line. Interleukin-13 activation of suppressor of cytokine signaling 1 (SOCS1) and eotaxin-3 (CCL26) expression was also diminished. MicroRNA-31/microRNA-155 mimics also downregulated IL13RA1 in ex vivo human inflamed UC biopsies. We propose that miR-31 and miR-155 have an important role in limiting IL-13 signalling in UC disease

Identification of host miRNAs that may limit human rhinovirus replication

Taken together, our results suggest that pathological changes in microRNA expression, as already reported for asthma or chronic obstructive pulmonary disease have the potential to affect Rhinovirus replication and therefore may play a role in virus-induced exacerbation

Copper transporters are responsible for copper isotopic fractionation in eukaryotic cells

International audienceCopper isotopic composition is altered in cancerous compared to healthy tissues. However, therationale for this difference is yet unknown. As a model of Cu isotopic fractionation, we monitored Cuuptake in Saccharomyces cerevisiae, whose Cu import is similar to human. Wild type cells are enrichedin 63Cu relative to 65Cu. Likewise, 63Cu isotope enrichment in cells without high-affinity Cu transportersis of slightly lower magnitude. In cells with compromised Cu reductase activity, however, no isotopefractionation is observed and when Cu is provided solely in reduced form for this strain, copper isenriched in 63Cu like in the case of the wild type. Our results demonstrate that Cu isotope fractionationis generated by membrane importers and that its amplitude is modulated by Cu reduction. Based on abinitio calculations, we propose that the fractionation may be due to Cu binding with sulfur-rich aminoacids: methionine and cysteine. In hepatocellular carcinoma (HCC), lower expression of the STEAP3copper reductase and heavy Cu isotope enrichment have been reported for the tumor mass, relative tothe surrounding tissue. Our study suggests that copper isotope fractionation observed in HCC could bedue to lower reductase activity in the tumor

A microRNA network dysregulated in asthma controls IL-6 production in bronchial epithelial cells

MicroRNAs are short non-coding single stranded RNAs that regulate gene expression. While much is known about the effects of individual microRNAs, there is now growing evidence that they can work in co-operative networks. MicroRNAs are known to be dysregulated in many diseases and affect pathways involved in the pathology. We investigated dysregulation of microRNA networks using asthma as the disease model. Asthma is a chronic inflammatory disease of the airways characterized by bronchial hyperresponsiveness and airway remodelling. The airway epithelium is a major contributor to asthma pathology and has been shown to produce an excess of inflammatory and pro-remodelling cytokines such as TGF-?, IL-6 and IL-8 as well as deficient amounts of anti-viral interferons. After performing microRNA arrays, we found that microRNAs -18a, -27a, -128 and -155 are down-regulated in asthmatic bronchial epithelial cells, compared to cells from healthy donors. Interestingly, these microRNAs are predicted in silico to target several components of the TGF-?, IL-6, IL-8 and interferons pathways. Manipulation of the levels of individual microRNAs in bronchial epithelial cells did not have an effect on any of these pathways. Importantly, knock-down of the network of microRNAs miR-18a, -27a, -128 and -155 led to a significant increase of IL-8 and IL-6 expression. Interestingly, despite strong in silico predictions, down-regulation of the pool of microRNAs did not have an effect on the TGF-? and Interferon pathways. In conclusion, using both bioinformatics and experimental tools we found a highly relevant potential role for microRNA dysregulation in the control of IL-6 and IL-8 expression in asthma. Our results suggest that microRNAs may have different roles depending on the presence of other microRNAs. Thus, interpretation of in silico analysis of microRNA function should be confirmed experimentally in the relevant cellular context taking into account interactions with other microRNAs when studying diseas

Copper isotope effect in serum of cancer patients. A pilot study

The isotope effect describes mass-dependent variations of natural isotope abundances for a particular element. In this pilot study, we measured the Cu-65/Cu-63 ratios in the serums of 20 breast and 8 colorectal cancer patients, which correspond to, respectively, 90 and 49 samples taken at different times with molecular biomarker documentation. Copper isotope compositions were determined by multiple-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). When compared with the literature data from a control group of 50 healthy blood donors, abundances of Cu isotopes predict mortality in the colorectal cancer group with a probability p = 0.018. For the breast cancer patients and the group of control women the probability goes down to p = 0.0006 and the AUC under the ROC curve is 0.75. Most patients considered in this preliminary study and with serum delta Cu-65 lower than the threshold value of -0.35 parts per thousand (per mil) did not survive. As a marker, a drop in delta Cu-65 precedes molecular biomarkers by several months. The observed decrease of delta Cu-65 in the serum of cancer patients is assigned to the extensive oxidative chelation of copper by cytosolic lactate. The potential of Cu isotope variability as a new diagnostic tool for breast and colorectal cancer seems strong. Shifts in Cu isotope compositions fingerprint cytosolic Cu chelation by lactate mono-and bidentates. This simple scheme provides a straightforward explanation for isotopically light Cu in the serum and isotopically heavy Cu in cancer cells: Cu+ escaping chelation by lactate and excreted into the blood stream is isotopically light. Low delta Cu-65 values in serum therefore reveal the strength of lactate production by the Warburg effect

Hypoxia induces copper stable isotope fractionation in hepatocellular carcinoma, in a HIF-independent manner

International audienc

MicroRNA-31 targets thymic stromal lymphopoietin in mucosal infiltrated CD4+ T cells: : a role in achieving mucosal healing in ulcerative colitis?

Background: Ulcerative colitis (UC) is characterized by disruption of the mucosal intestinal barrier. MicroRNAs, single-stranded noncoding RNAs of approximately 22nt, are dysregulated in UC. MicroRNAs targeting thymic stromal lymphopoietin (TSLP), a cytokine involved in T-cell maturation and polarization, may be involved in regulating UC inflammation and mucosal healing.Methods: Biopsy samples from non-UC (n = 38), inactive UC (n = 18), and active UC (n = 23) patients were analyzed for mRNA (real-time quantitative polymerase chain reaction) or TSLP protein expression (enzyme-linked immunosorbent assay). Flow cytometry was used to isolate CD4+ T cells from biopsies. The functional mechanism was shown using luciferase assays and antago-miR transfections. The TSLP/miR-31 association was analyzed on 196 subjects from a previous clinical trial that tested the anti-IL-13 drug tralokinumab, whereas mucosal healing effects were studied on a subset of patients (n = 13) from this trial.Results: We found that TSLP is reduced at both mRNA and protein levels in inflamed UC patients when compared with healthy subjects, in both whole biopsies and biopsy-isolated CD4+ CD25+ T cells. The expression of miR-31, predicted to target TSLP, inversely co-related to the levels of TSLP mRNA in T cells. Blocking miR-31 in vitro in T cells increased both TSLP mRNA expression and protein secretion. Luciferase assays showed that miR-31 directly targeted TSLP mRNA, suggesting a direct mechanistic link. We also found that TSLP is increased in patients who achieve mucosal healing, comparing biopsies before and after treatment from the tralokinumab trial.Conclusions: Our data suggest a role for TSLP in promoting mucosal healing and regulating inflammation in UC, whereas miR-31 can directly block this effect

Sulfur isotope analysis by MC-ICP-MS and application to small medical samples

We describe a technique of S isotope analysis in sulfate form with the first separation stage involving anion-exchange and the second stage of mass-spectrometric analysis by MC-ICP-MS using standard-sample-standard bracketing. Ammonium in 1 : 1 stoichiometric proportion with sulfate was used to improve transmission and stability and to avoid cone and membrane clogging by condensable species. The working resolution of similar to 9000 allowed the main interferences, notably (SH)-S-32 on S-33, to be resolved. The matrix effect caused by phosphorus present in biological samples is negligible for S/P ratios \textgreater= 10: our chemical protocol allows S/P \textgreater= 150 to be routinely achieved. Replicate measurements of S standard solutions give values of isotopic abundances within errors of accepted values and demonstrate a reproducibility of +/- 0.10 parts per thousand for delta S-34 and +/- 0.15 parts per thousand for delta S-33 (2s). The technique is adequate for quantities as small as 10 nanomoles. We investigated the delta S-34 of 110 samples of cancer patients and 10 samples of rheumatoid arthritis patients. We avoided the use of blood collection tubes with sulfate-containing heparin. Sulfur in serum is transported by albumin and fibrinogen. Most serum and plasma delta S-34 values fall within a narrow interval of similar to 1 parts per thousand around a mean delta S-34(VCDT) of similar to 6.0 parts per thousand. The delta S-34 values of total blood, serum, and plasma are very similar. Despite the short turnover time of albumin and fibrinogen, S is surprisingly well regulated. Subtle variations of 0.2-0.3 parts per thousand around the mean value can be assigned to sex and age, with sulfur in male and adult samples tending to be heavier than in their female and juvenile counterparts. This narrow range of variations across the spectrum of a large number of individuals not selected for controlled dietary habits seems paradoxical. In general, breast and prostate cancer and rheumatoid arthritis have very little effect on the average serum delta S-34, but increase the scatter of values. We confirm that the serum of patients affected by liver cancer and other pathologies is depleted of albumin-born sulfur. While sulfur in the serum of patients with non-malignant liver pathologies tends to be isotopically light, the serum delta S-34 of medicated hepatocellular carcinoma patients tends to be at the high end of control values

MiR-27a and miR-128 directly target the 3′UTR of SMAD2.

<p><i>A</i> Renilla luciferase construct harbouring an SMAD2 3′-UTR fragment containing the predicted binding sites for miR-27a and miR-128 (wild type, WT) or its mutated version for both miRNAs binding (MUT) was co-transfected with either an empty expression vector, a miR-27a over-expressing vector (WT+27a and MUT+27a, respectively) or an miR-128 over-expressing vector (WT+128 and MUT+miR-128, respectively. One of three independent experiments is shown. Where no significance is shown = non-significant, ***p≤0.001. Error bars indicate Standard Error. <i>B</i> Schematic representing the binding region (and mutation site) of miR-27 and miR-128 in SMAD2 3′UTR.</p