Magnetic Resonance Enterography and Histology in Patients With Fibrostenotic Crohn's Disease: A Multicenter Study

INTRODUCTION Magnetic resonance enterography (MRE) is useful for detecting bowel strictures, whereas a number of imaging biomarkers may reflect severity of fibrosis burden in Crohn's disease (CD). This study aimed to verify the association of MRE metrics with histologic fibrosis independent of inflammation. METHODS This prospective European multicenter study performed MRE imaging on 60 patients with CD with bowel strictures before surgical resection. Locations of 61 histological samples were annotated on MRE examinations, followed by central readings using the Chiorean score and measurement of delayed gain of enhancement (DGE), magnetization transfer ratio, T2-weighted MRI sequences (T2R), apparent diffusion coefficient (ADC), and the magnetic resonance index of activity (MaRIA). Correlations of histology and MRE metrics were assessed. Least Absolute Shrinkage and Selection Operator and receiver operator characteristic (ROC) curve analyses were used to select composite MRE scores predictive of histology and to estimate their predictive value. RESULTS ADC and MaRIA correlated with fibrosis (R = -0.71, P < 0.0001, and 0.59, P < 0.001) and more moderately with inflammation (R = -0.35, P < 0.01, and R = 0.53, P < 0.001). Lower or no correlations of fibrosis or inflammation were found with DGE, magnetization transfer ratio, or T2R. Least Absolute Shrinkage and Selection Operator and ROC identified a composite score of MaRIA, ADC, and DGE as a very good predictor of histologic fibrosis (ROC area under the curve = 0.910). MaRIA alone was the best predictor of histologic inflammation with excellent performance in identifying active histologic inflammation (ROC area under the curve = 0.966). DISCUSSION MRE-based scores for histologic fibrosis and inflammation may assist in the characterization of CD stenosis and enable development of fibrosis-targeted therapies and clinical treatment of stenotic patients

Model of the role of miR-375 in the regulation of apoptotic death.

<p>Model of the role of miR-375 in the regulation of apoptotic death.</p

mRNA profiling of HCT116 cells upon ectopic expression of miR-375 and miR-375 target identification.

<p>(A) Reconstitution of mature miR-375 upon transfection with pre-miR-375 or Scr (RT-qPCR). (B) Cumulative fraction plotted as a function of log2 fold changes. The mRNAs were dichotomized according to the presence or absence of minimum one seed match in the 3′UTR or according to target prediction using Target Scan v5.2. The mRNAs with minimum one 7mer-m8 seed match within the 3′ UTR showed a higher propensity to down-regulation upon miR-375 over-expression. (C) The mRNAs were ranked according to fold change and grouped into a total of 23 bins. Upper panel: The average 7mer-8m seed frequency within the 3′UTR regions in each bin was calculated. Bottom panel: Overall miRNA-induced mRNA fold change. (D and E) Relative expression of HELLS, NOLC1, YAP1, BIRC5 and BCL2L1 upon ectopic miR-375 expression using RT-qPCR. (F) Western blots demonstrating the effect of miR-375 on the protein level of HELLS, NOLC1 and YAP1 in HCT116 cells. Loading control: β-actin. *<i>p</i><0.05. (G–H) Ago 2 immunoprecipitation. (G) RT-qPCR expression analysis of YAP1 in the cell lysates of miR-375 or Scr transfected cells (input) used for Ago2 immunoprecipitation. (H) Ago2 immunoprecipitation from cell lysates of miR-375 or Scr transfected cells (IP) followed by YAP1 expression analysis using RT-qPCR. Immunoprecipitation with a FLAG antibody was used as negative control. A 1∶1 ratio of the lysates from miR-375 and Scr transfected cells was used for FLAG immunoprecipitation. The columns represent the mean of 3 replicates ± sd.</p

The most differentially expressed miRNAs in stage II colorectal adenocarcinomas and their ability to induce phenotypic changes in the high-throughput analysis.

1<p>A FC _(log2)≤−1.50 and ≥1.50 and a p-value≤0.01 was considered significant (Mann-Whitney U test).</p><p>NA: miRNAs not included in the pre-miRNA library from Ambion.</p><p>+: miRNAs that induced phenotypic changes (Top-40 ranked).</p><p>(+): miRNAs that induced phenotypic changes in at least one cell line (not Top-40 ranked).</p><p>A: Induction of apoptosis.</p><p>P: Inhibition of proliferation.</p

Expression of known direct miR-375 targets.

Δ<p>Analyzed in 24 normal mucosa and 30 MSS adenocarcinomas that has previously been profiled using Human Exon 1.0 ST arrays (Thorsen K et al. Alternative Splicing of SLC39A14 in Colorectal Cancer is Regulated by the Wnt Pathway,</p><p>Molecular and Cellular Proteomics, 2011).</p><p>ND: not detected (median log intensity <7).</p><p>NS: not significant (a p-value≤0.05 was considered significant).</p>‡<p>: not present on the Human Gene 1.0ST.</p><p>*Hs: Homo sapiens.</p>¤<p>Mm: Mus musculus.</p>∧<p>The probes on the Human Gene 1.0ST arrays recognize more than one transcript.</p

Generation and characterization of stable HCT116 cells with inducible miR-375 expression (HCT116-miR-375H).

<p>(A) Dox treatment induces increased expression of miR-375 in HCT116_miR-375H cells. The Relative expression of miR-375 was measured using RT-qPCR. The columns represent the mean of 3 replicates ± sd.*p-value<0.05 when compared to HCT116_ScrH cells. (B) xCELLigence real-time monitoring of cell proliferation. The cell index from time 12–96 hours is shown. Dox was added at time 0. (C) Dox treatment significantly reduces the proliferation of HCT116_miR-375H cells. Following the real-time monitoring in B, the slope (rate of changes in cell index) was calculated from time 60–80 hours (i.e. when changes in cell viability were apparent) and presented graphically. (D) Dox treatment specifically induces Caspase 3/7 dependent apoptosis in HCT116 miR-375H cells. The Caspase 3/7 activity was examined by fluorometric kinetic analysis and expressed relative to the Caspase 3/7 activity in untreated HCT116_ScrH cells. Z-DEVD-fmk (DEVD) was added to the cells six hours post-transfection. Data are presented as ±sd. of at least 2 independent experiments each with three biological replicates. *p-value<0.05. (E) Western blotting demonstrating down-regulation of YAP1 in dox treated HCT116_miR-375H cells compared to non-treated and HCT116_ScrH cells. Loading control: β-actin. (F) miR-375 expression reduces tumor growth <i>in vivo</i>. Growth curves of tumors generated in nude mice injected with HCT116_miR-375H cells treated with (n = 4) or without (n = 4) dox in the drinking water. Dox was added to the drinking water when the tumor size was >50 mm³. Data marks and bars represent the mean ±sd. *p-value<0.05.</p