ABSTRACT
We observed persistent peritoneal bacteria despite a transient early innate immune response to intraperitoneal (IP) Klebsiella pneumoniae. Pretreatment with LPS prior to peritonitis induced a tolerant pattern of pro-inflammatory cytokine protein production over 72 h, but not at the mRNA level. MicroRNAs (miRNAs) regulate inflammatory cytokines and may explain this paradox. After pretreatment with IP LPS or saline, C57BL/6 mice were given 10(3) CFU of K. pneumoniae IP. Total RNA was isolated from peritoneal exudate cells (4 h, 24 h and 48 h following infection). mRNA and miRNA expression levels were detected and bioinformatics pathway analysis was performed, followed by measuring TNF-α, IL-1ß, IL-6 and High-mobility Group Box 1 (HMBG1) protein levels. Of 88 miRNAs studied, 30 were significantly dysregulated at all time points in the LPS-pretreated group, including MiR-155, -146a, -142-3p, -299, and -200c -132 and -21. TNF-α, regulated by miR-155 and miR-146a, was decreased in the LPS-pretreated group at all time points (P < 0.05), as were HMGB1, a key alarmin regulated by miR-146, -142-3p, -299 and -200c (P < 0.05), and IL-1ß and IL-6, both regulated by miR-132and miR-21 respectively (P < 0.05). Specific dysregulation of miR-155, -146a, -142-3p, -299, and -200c -132 and -21 with their corresponding effects on the TLR and NF-κB signaling pathways during inflammation, suggests a plausible mechanism for tolerance in this novel chronic model with persistent peritoneal infection.
Subject(s)
Klebsiella Infections/immunology , Klebsiella pneumoniae/immunology , MicroRNAs/metabolism , Peritonitis/immunology , RNA, Messenger/metabolism , Animals , Cells, Cultured , Chronic Disease , Computational Biology , Cytokines/metabolism , Disease Models, Animal , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Humans , Immune Tolerance , Inflammation Mediators/metabolism , Klebsiella Infections/complications , Male , Mice , Mice, Inbred C57BL , MicroRNAs/genetics , MicroRNAs/immunology , NF-kappa B/metabolism , Peritonitis/etiology , RNA, Messenger/genetics , RNA, Messenger/immunology , Repressor Proteins/genetics , Repressor Proteins/metabolism , Signal Transduction/immunologyABSTRACT
OBJECTIVES: The main objective of this study was to investigate the potential use of circulating microRNAs (miRNAs) as biomarkers of sporadic colorectal cancer (CRC). BACKGROUND: CRC, a leading cause of death, is curable if detected early. There is an unmet need for an accurate, noninvasive biomarker of CRC. MiRNAs are non-protein-coding RNAs regulating gene expression that play a role in CRC development. METHODS: Levels of 380 miRNAs were determined using microfluidic array technology (Applied Biosystems) in a "training" set of 30 CRC patients from whom cancer and adjacent normal tissue were collected. The 4 most dysregulated miRNAs (P < 0.05, false discovery rate (FDR): 10%) were then validated in a second blinded "test" set of 16 CRC patients from whom cancer and normal adjacent tissue had been collected. Validated tissue miRNAs were then evaluated in a plasma "test" set consisting of 30 CRC patients and 30 individuals without CRC. The most dysregulated tissue miRNAs were then validated in an independent new plasma test set consisting of 20 CRC patients with 20 age-, -, and race-matched subjects without CRC. RESULTS: Nineteen of 380 miRNAs were dysregulated in CRC tissue in the tissue "training" set (P < 0.05, FDR: 10%). The 2 most upregulated (miR-31; miR-135b) and most downregulated (miR-1; miR-133a) miRNAs identified CRC in our "test" set with 100% sensitivity and 80% specificity. MiR-31 was more upregulated in stages III and IV compared with stages I and II (P < 0.05). In the "plasma" group, miR-21 differentiated CRC patients from controls with 90% specificity and sensitivity. CONCLUSIONS: Plasma miRNAs provide reliable and noninvasive markers for CRC. Plasma miR-21 warrants study in larger cohorts. It seems uniquely promising as a plasma biomarker for CRC.
Subject(s)
Biomarkers, Tumor/metabolism , Colorectal Neoplasms/diagnosis , MicroRNAs/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/blood , Case-Control Studies , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Down-Regulation , Female , Genetic Markers , Humans , Male , MicroRNAs/blood , Middle Aged , Neoplasm Staging , Sensitivity and Specificity , Up-RegulationABSTRACT
One of the most serious complications faced by patients with inflammatory bowel disease (IBD) is the potential development of colorectal cancer (CRC). There is a compelling need to enhance the accuracy of cancer screening of IBD patients. MicroRNAs (miRNAs) are small nonprotein-coding RNAs that play important roles in CRC oncogenesis. In this study, we report differential miRNA expression in IBD patients with associated CRC from non-neoplastic tissue to dysplasia and eventually cancer. In addition, we identify and examine the role of dysregulated miRNAs in the TP53 pathway. In our CD patients, six miRNAs were upregulated from non-neoplastic tissue to dysplasia, but downregulated from dysplasia to cancer (miR-122, miR-181a, miR-146b-5p, let-7e, miR-17, miR-143) (P < 0.001). Six differentially expressed miRNAs affected the TP53 pathway (miR-122, miR-214, miR-372, miR-15b, let-7e, miR-17) (P < 0.001). Using two human colon cancer cell lines (HT-29 and HCT-116), E2F1, an upstream regulator of TP53, was downregulated in both cell lines transfected with let-7e (P < 0.05) as well as in HCT-116 cells transfected with miR-17 (P < 0.05). Additionally, cyclin G, a cell-cycle regulator miR-122 target was downregulated in both cell lines (P < 0.05). Unique differentially expressed miRNAs were observed in CD-associated CRC progression. Six of these miRNAs had a tumorigenic effect on the TP53 pathway; the effect of three of which was studied using cell lines.