Data_Sheet_1_Induction of Secretagogue Independent Gastric Acid Secretion via a Novel Aspirin-Activated Pathway.PDF

Aspirin has been widely recommended for acute and chronic conditions for over 2,000 years. Either single or repetitive doses are commonly used for analgesic and antipyretic reasons and to prevent heart attacks, stroke, and blood clot formation. Recent studies show that it can also be used chronically to dramatically reduce the risk of a variety of cancers. However, prolonged usage of aspirin can cause severe damage to the mucosal barrier, increasing the risk of ulcer formation and GI-bleeding events. In the present study, we show the effects of acute low-dose aspirin exposure as an active secretagogue-inducing gastric acid secretion. Studies were carried out with isolated gastric glands using the pH-sensitive dye BCECF-AM to assess acid secretion. The non-selective NOS inhibitor L-NAME (30 μM), or the specific inhibitor ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) was applied while monitoring intracellular pH. The effects of basolateral exposure to aspirin (acetylsalicylic acid, ASA) caused activation of gastric acid secretion via the H+, K+-ATPase. Our data suggest that aspirin increases nitric oxide (NO) production, which in turn activates acid secretion. Exposure of gastric glands to either the non-selective NOS inhibitor L-NAME, and the highly selective, soluble guanylyl cyclase inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) effectively inhibited aspirin-dependent gastric acid secretion. Aspirin can be considered as a novel secretagogue, in the way that it activates the H+, K+-ATPase. With increased daily aspirin consumption, our findings have important implications for all individuals consuming aspirin even in low doses and the potential risks for increased acid secretion.</p

Feasibility of Fecal MicroRNAs as Novel Biomarkers for Pancreatic Cancer

<div><h3>Introduction</h3><p>Pancreatic cancer (PCA) is an aggressive tumor that associates with high mortality rates. Majority of PCA patients are diagnosed usually at late tumor stages when the therapeutic options are limited. MicroRNAs (miRNA) are involved in tumor development and are commonly dysregulated in PCA. As a proof-of-principle study, we aimed to evaluate the potential of fecal miRNAs as biomarkers for pancreatic cancer.</p> <h3>Materials and Methods</h3><p>Total RNA was extracted from feces using Qiagen's miRNA Mini Kit. For miRNA expression analyses we selected a subset of 7 miRNAs that are frequently dysregulated in PCA (miR-21, -143, -155, -196a, -210, -216a, -375). Subsequently, expression levels of these miRNAs were determined in fecal samples from controls (n = 15), chronic pancreatitis (n = 15) and PCA patients (n = 15) using quantitative TaqMan-PCR assays.</p> <h3>Results</h3><p>All selected miRNAs were detectable in fecal samples with high reproducibility. Four of seven miRNAs (miR-216a, -196a, -143 und -155) were detected at lower concentrations in feces of PCA patients when compared to controls (p<0.05). Analysis of fecal miRNA expression in controls and patients with chronic pancreatitis and PCA revealed that the expression of miR-216a, -196a, -143 und -155 were highest in controls and lowest in PCA. The expression of the remaining three miRNAs (miR-21, -210 and -375) remained unchanged among controls and the patients with either chronic pancreatitis or PCA.</p> <h3>Conclusion</h3><p>Our data provide novel evidence for the differential expression of miRNAs in feces of patients with PCA. If successfully validated in large-scale prospective studies, the fecal miRNA biomarkers may offer novel tools for PCA screening research.</p> </div

Stability of fecal miRNAs.

<p>To evaluate the long-term stability of the samples, we performed miRNA analyses in the fecal samples collected at different time points. Samples 1–15 were collected between 2004 and 2006 and samples 16–30 between 2009 and 2010 from healthy subjects. All samples were stored and processed in similar conditions. Figure (A) shows variations in miR-16 and miR-196a expression among all feces samples. (B) miR-16 and (C) miR-196a expression in subgroup analyses showed similar expression (p>0.1). (D) Normalized miR-196a expression is comparable in long- and short-term stored samples (p = 0.441). (E) Since miR-216a was present in feces at lowest concentrations, and its expression was analyzed by two independent quantitative RT-PCR runs to evaluate the reproducibility of the analysis (p<0.0001). Normalization was performed with using miR-16 as internal normalizer.</p

Clinico-pathological characteristics of patients included to the study.

*<p> <i>- at time point of sample collection;</i></p>#<p> <i>- endocrine pancreatic insufficiency is defined by impaired glucose tolerance test or by manifest diabetes mellitus;</i></p>§<p>- exocrine pancreatic insufficiency is defined by reduced elastase in stool.</p

Cumulative miRNA expression analyses improve the separation of PCA samples.

<p>Summation of the ΔCt-values of miR-196a, -216a, -143 and -155 was performed to calculate the Σ ΔCt-value. *- p<0.05, ***- p<0.001. Abbreviations: N-control subjects, CP- chronic pancreatitis, PCA- pancreatic cancer. The data are present as box-and-whiskers plots: the upper and lower limits of the boxes indicate the 75^th and 25^th percentiles, the lines inside the boxes - the medians, and the upper and lower horizontal bars denote the 90^th and 10^th percentiles, respectively.</p

JCV miRNA sequence and detection.

<p>(A) Schematic presentation of the JCV genome. The black circle marks the transcript location of the JCV miR-J1 stem loop. (B) JCV miR-J1-5p and -3p sequences are compared to the Merkel Cell Polyomavirus-, SV40- and BK virus-miRNA sequences. (C & D) CRC cells were transfected in vitro with a JCVT-Ag-E plasmid, and JCV T-Ag message and miRNA expression were analyzed. In C, GAPDH and β-actin were used as loading controls for mRNA and protein expression, respectively. (D) Vector transfected cells showed no detectable miR-J1-5p expression, while JCV miR-J1-5p expression was high in transfected cells. To measure the expression of miR-J1-5p, expression in the vector was set to a Ct-value of 40, and 2^−ΔΔCt values were calculated using RNU6b for normalization.</p

The expression of analyzed fecal microRNAs in comparison to tumor tissues, pancreatic fluid and blood of patients with pancreatic cancer.

<p>The expression of analyzed fecal microRNAs in comparison to tumor tissues, pancreatic fluid and blood of patients with pancreatic cancer.</p

MiRNA expression patterns in patients with chronic pancreatitis and PCA.

<p>Figures (A) to (G) represent different miRNAs that were selected for the study based on the alterations in PCA tissues. *represents p<0.05, ***- p<0.001. Abbreviations: N-control subjects, CP- chronic pancreatitis, PCA- pancreatic cancer. The data are present as box-and-whiskers plots: the upper and lower limits of the boxes indicate the 75^th and 25^th percentiles, the lines inside the boxes - the medians, and the upper and lower horizontal bars denote the 90^th and 10^th percentiles, respectively.</p

Detection of fecal miRNAs that are commonly dysregulated in pancreatic cancer tissues.

<p>(A) MiRNA microarray expression analyses were performed using Illumina microarray to evaluate the presence of selected miRNAs (miR-16, -375, -196a, -216a, -21, -143, -155 and -210) in a single stool sample of the healthy subject. Fecal miRNA expression was converted to log-expression values following Lumi Bioconductor normalization. (B & C) Expression of selected miRNA was confirmed in all 45 samples including controls, chronic pancreatitis and pancreatic cancer patients. Figure (B) represents the raw miRNA expression Ct-values for qualitative assessment. (C) Normalization was performed using standard ΔCt-method using miR-16 as internal fecal normalizer.</p

JCV miR-J1-5p detection in feces.

<p>(A) To test whether JCV miRNA is present in stool, we extracted total RNA from stool samples and performed TaqMan based miRNA expression analyses. Expression of miR-J1-5p was normalized to mean miR-16 and -26b levels and further adjusted to the sample with the lowest miR-J1-5p expression level (1*). (B) To test the reproducibility of miRNA detection, we performed independent RNA extraction from the same samples in the subset of fecal samples from healthy subjects (n = 5). The samples were normalized to mean miR-16 and -26b expression. (C) Concomitant expression analyses of miR-J1-5p and -3p showed no correlation with JCV miRNA expression, arguing for potential cross-reactivity with BKV microRNA. (D&E) To measure JCV miR-J1-5p expression in feces from CRC patients, miR-J1-5p was analyzed by TaqMan PCR in 29 FOBT specimens from patients without and with colorectal neoplasia. Fold-expression was calculated using the 2^−ΔCt method normalized to mean miR-16 and -26b expression. D Represents the single sample values and E the mean values ± SD.</p