Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples

Additional file 1: Table S1. Taxonomic listing of all bacterial genera and species detected as contaminants of DNA extraction and processing kits in the present study and previously reported

Blood-based microRNAs as biomarkers for the diagnosis of colorectal cancer: a systematic review and meta-analysis

Background: Colorectal cancer (CRC) is common and associated with significant mortality. Current screening methods for CRC lack patient compliance. microRNAs (miRNAs), identified in body fluids, are negative regulators of gene expression and are dysregulated in many cancers, including CRC. This paper summarises studies identifying blood-based miRNAs dysregulated in CRC compared with healthy controls in an attempt to evaluate their use as a screening tool for the diagnosis of CRC. Methods: A search of electronic databases (PubMed and EMBASE) and grey literature was performed between January 2002 and April 2016. Studies reporting plasma or serum miRNAs in the diagnosis of CRC compared with healthy controls were selected. Patient demographics, type of patient sample (serum or plasma), method of miRNA detection, type of normalisation, and the number of significantly dysregulated miRNAs identified were recorded. Statistical evaluation of dysregulated miRNAs using sensitivity, specificity, and area under the curve (AUC) was performed. Results: Thirty-four studies investigating plasma or serum miRNAs in the diagnosis of CRC were included. A total of 31 miRNAs were found to be either upregulated (n=17) or downregulated (n=14) in CRC cases as compared with controls. Fourteen studies identified panels of ⩾2 dysregulated miRNAs. The highest AUC, 0.943, was identified using a panel of 4 miRNAs with 83.3% sensitivity and 93.1% specificity. Meta-analysis of studies identifying a single dysregulated miRNA in CRC cases compared with controls was performed. Overall sensitivity and specificity of 28 individual miRNAs in the diagnosis of CRC were 76% (95% CI 72%–80%) and 76% (95% CI 72%–80%), respectively, indicating good discriminative ability of miRNAs as biomarkers for CRC. These data did not change with sensitivity analyses. Conclusions: Blood-based miRNAs distinguish patients with CRC from healthy controls with high sensitivity and specificity comparable to other common and invasive currently used screening methods for CRC. In future, miRNAs may be used as a relatively non-invasive blood-based marker for detection of CRC

Long Non-Coding RNA ZFAS1 Is a Major Regulator of Epithelial-Mesenchymal Transition through miR-200/ZEB1/E-Cadherin, Vimentin Signaling in Colon Adenocarcinoma

Colon adenocarcinoma is a common cause of cancer-related deaths worldwide. Epithelial-mesenchymal transition is a major regulator of cancer metastasis, and increased understanding of this process is essential to improve patient outcomes. Long non-coding RNA (lncRNA) are important regulators of carcinogenesis. To identify lncRNAs associated with colon carcinogenesis, we performed an exploratory differential gene expression analysis comparing paired colon adenocarcinoma and normal colon epithelium using an RNA-sequencing data set. This analysis identified lncRNA ZFAS1 as significantly increased in colon cancer compared to normal colon epithelium. This finding was validated in an institutional cohort using laser capture microdissection. ZFAS1 was also found to be principally located in the cellular cytoplasm. ZFAS1 knockdown was associated with decreased cellular proliferation, migration, and invasion in two colon cancer cell lines (HT29 and SW480). MicroRNA-200b and microRNA-200c (miR-200b and miR-200c) are experimentally validated targets of ZFAS1, and this interaction was confirmed using reciprocal gene knockdown. ZFAS1 knockdown regulated ZEB1 gene expression and downstream targets E-cadherin and vimentin. Knockdown of miR-200b or miR-200c reversed the effect of ZFAS1 knockdown in the ZEB1/E-cadherin, vimentin signaling cascade, and the effects of cellular migration and invasion, but not cellular proliferation. ZFAS1 knockdown was also associated with decreased tumor growth in an in vivo mouse model. These results demonstrate the critical importance of ZFAS1 as a regulator of the miR-200/ZEB1/E-cadherin, vimentin signaling cascade

The microRNA‑200 family acts as an oncogene in colorectal cancer by inhibiting the tumor suppressor RASSF2

This study aimed to determine whether manipulation of the microRNA‑200 (miR‑200) family could influence colon adenocarcinoma cell behavior. The miR‑200 family has a significant role in tumor suppression and functions as an oncogene. In vitro studies on gain and loss of function with small interfering RNA demonstrated that the miR‑200 family could regulate RASSF2 expression. Knockdown of the miR‑200 family in the HT‑29 colon cancer cell line increased KRAS expression but decreased signaling in the MAPK/ERK signaling pathway through reduced ERK phosphorylation. Increased expression of the miR‑200 family in the CCD‑841 colon epithelium cell line increased KRAS expression and led to increased signaling in the MAPK/ERK signaling pathway but increased ERK phosphorylation. Functionally, knockdown of the miR‑200 family led to decreased cell proliferation in the HT‑29 cells; therefore, increased miR‑200 family expression could increase cell proliferation in the CCD‑841 cell line. The present study included a large paired miR array dataset (n=632), in which the miR‑200 family was significantly found to be increased in colon cancer when compared with normal adjacent colon epithelium. In a miR‑seq dataset (n=199), the study found that miR‑200 family expression was increased in localized colon cancer compared with metastatic disease. Decreased expression was associated with poorer overall survival. The miR‑200 family directly targeted RASSF2 and was inversely correlated with RASSF2 expression (n=199, all P<0.001). Despite the well‑defined role of the miR‑200 family in tumor suppression, the present findings demonstrated a novel function of the miR‑200 family in tumor proliferation

Evaluation of SLC11A1 as an inflammatory bowel disease candidate gene

BACKGROUND: Significant evidence suggests that a promoter polymorphism withinthe gene SLC11A1 is involved in susceptibility to both autoimmune and infectious disorders. The aim of this study was to evaluate whether SLC11A1 has a role in the susceptibility to inflammatory bowel disease (IBD) by characterizing a promoter polymorphism within the gene and two short tandem repeat (STR) markers in genetic proximity to SLC11A1. METHODS: The studied population consisted of 484 Caucasians with IBD, 144 population controls, and 348 non-IBD-affected first-degree relatives of IBD patients. IBD subjects were re-categorized at the sub-disease phenotypic level to characterize possible SLC11A1 genotype-phenotype correlations. Polymorphic markers were amplified from germline DNA and typed using gel electrophoresis. Genotype-phenotype correlations were defined using case-control, haplotype, and family-based association studies. RESULTS: This study did not provide compelling evidence for SLC11A1 disease association; most significantly, there was no apparent evidence of SLC11A1 promoter allele association in the studied Crohn's disease population. CONCLUSION: Our results therefore refute previous studies that have shown SLC11A1 promoter polymorphisms are involved in susceptibility to this form of IBD

The role and function of IκKα/β in monocyte impairment

Following major trauma, sepsis or surgery, some patients exhibit an impaired monocyte inflammatory response that is characterized by a decreased response to a subsequent bacterial challenge. To investigate this poorly understood phenomenon, we adopted an in-vitro model of endotoxin tolerance utilising primary human CD14 + monocytes to focus on the effect of impairment on IκKα/β, a critical part of the NFκB pathway. Impaired monocytes had decreased IκKα mRNA and protein expression and decreased phosphorylation of the IκKα/β complex. The impaired monocyte secretome demonstrated a distinct cytokine/chemokine footprint from the naïve monocyte, and that TNF-α was the most sensitive cytokine or chemokine in this setting of impairment. Inhibition of IκKα/β with a novel selective inhibitor reproduced the impaired monocyte phenotype with decreased production of TNF-α, IL-6, IL-12p70, IL-10, GM-CSF, VEGF, MIP-1β, TNF-β, IFN-α2 and IL-7 in response to an LPS challenge. Surgical patients with infection also exhibited an impaired monocyte phenotype and had decreased SITPEC, TAK1 and MEKK gene expression, which are important for IκKα/β activation. Our results emphasize that impaired monocyte function is, at least in part, related to dysregulated IκKα/β activation, and that IκKα/β is likely involved in mounting a sufficient monocyte inflammatory response. Future studies may wish to focus on adjuvant therapies that augment IκKα/β function to restore monocyte function in this clinically important problem

Clinical predictors of inflammatory bowel disease in a genetically well-defined Caucasian population

<p>Abstract</p> <p>Background</p> <p>Crohn's disease (CD) and ulcerative colitis (UC), the two main types of inflammatory bowel disease (IBD), are multifactorial conditions of unknown etiology. The objective of this study is to examine the combined gene-environment interactions influencing IBD susceptibility in a well-defined Caucasian cohort in rural mid-America.</p> <p>Methods</p> <p>Patients were diagnosed to have CD or UC using conventional radiologic, endoscopic, and/or histopathologic findings. Histological diagnosis was made by a single specialist gastrointestinal pathologist with a particular interest in IBD. Information regarding cigarette smoke exposure was obtained by administration of the Behavioral Risk Factor Surveillance System Survey (BRFSS) to all patients. Genomic DNA was extracted from peripheral blood leukocytes, and polymerase chain reaction (PCR) amplification and genotyping were performed for 11 Single Nucleotide Polymorphisms (SNP) in <it>NOD2</it>, <it>IL23r</it>, <it>OCTN1 </it>genes along with <it>IGR</it>.</p> <p>Results</p> <p>Our cohort consists of 1196 patients: 435 controls, 485 CD patients, and 276 UC patients. Only patients with genotype data for at least 7 of 11 SNPs were included in our data analysis. The control groups for all 11 SNPs were in Hardy-Weinberg Equilibrium. In genotype-association SNP analysis, all <it>NOD2 </it>SNPs (rs5743293, rs2066844, rs2066845) and the <it>IL23r </it>SNP (rs11465804) showed a significant association to IBD (<it>p </it>< 0.03). A multiple gene-interaction analysis showed an association between <it>NOD2 </it>and <it>IL23r </it>with UC (<it>p </it>= 0.04). There were no associations between any <it>OCTN1 </it>and <it>IGR </it>SNPs and IBD in this cohort. A multivariable logistic regression analysis showed that female gender, "current" or "former" smoking status, family history of IBD, and <it>NOD2 </it>SNP minor alleles were associated with CD.</p> <p>Conclusion</p> <p>IBD remains to be challenging to properly diagnose, characterize, and treat. Our study proposes a combined genetic, phenotypic, and environmental approach in an attempt to better understand IBD. Previously demonstrated associations between OCTN1 and IGR and IBD were not confirmed.</p

Characterization of N-acetyltransferase 1 and 2 polymorphisms and haplotype analysis for inflammatory bowel disease and sporadic colorectal carcinoma

<p>Abstract</p> <p>Background</p> <p>N-acetyltransferase 1 (NAT1) and 2 (NAT2) are polymorphic isoenzymes responsible for the metabolism of numerous drugs and carcinogens. Acetylation catalyzed by NAT1 and NAT2 are important in metabolic activation of arylamines to electrophilic intermediates that initiate carcinogenesis. Inflammatory bowel diseases (IBD) consist of Crohn's disease (CD) and ulcerative colitis (UC), both are associated with increased colorectal cancer (CRC) risk. We hypothesized that <it>NAT1 </it>and/or <it>NAT2 </it>polymorphisms contribute to the increased cancer evident in IBD.</p> <p>Methods</p> <p>A case control study was performed with 729 Caucasian participants, 123 CRC, 201 CD, 167 UC, 15 IBD dysplasia/cancer and 223 controls. <it>NAT1 </it>and <it>NAT2 </it>genotyping were performed using Taqman based techniques. Eight single nucleotide polymorphisms (SNPs) were characterized for <it>NAT1 </it>and 7 SNPs for <it>NAT2</it>. Haplotype frequencies were estimated using an Expectation-Maximization (EM) method. Disease groups were compared to a control group for the frequencies at each individual SNP separately. The same groups were compared for the frequencies of <it>NAT1 </it>and <it>NAT2 </it>haplotypes and deduced NAT2 phenotypes.</p> <p>Results</p> <p>No statistically significant differences were found for any comparison. Strong linkage disequilibrium was present among both the <it>NAT1 </it>SNPs and the <it>NAT2 </it>SNPs.</p> <p>Conclusion</p> <p>This study did not demonstrate an association between <it>NAT1 </it>and <it>NAT2 </it>polymorphisms and IBD or sporadic CRC, although power calculations indicate this study had sufficient sample size to detect differences in frequency as small as 0.05 to 0.15 depending on SNP or haplotype.</p