Alcoholic Liver Disease (ALD) has high morbidity and mortality with no satisfactory therapy. Since endotoxemia is an essential co-factor for the development of ALD, we traced the origins of endotoxemia to leakiness of the intestinal barrier. How ethanol (EtOH) causes barrier hyperpermeability is unclear, but several lines of evidence suggest that it could be due to EtOH-induced dysregulation of tight junction proteins such as zonula occluden 1 (ZO-1), which are known to play important roles in regulation of intestinal permeability. MicroRNAs (miRNAs) are recently discovered small noncoding RNAs that can regulate gene expression by targeting mRNAs and triggering either translational repression or RNA degradation. ZO-1 is predicted to be a target gene of one such miRNA, miR-212. Accordingly, this proposal is designed to study the role of miR-212 in the mechanisms of alcohol-induced gut leakiness that is associated with ALD. Our preliminary data support a mechanistic role for miR-212 in EtOH-induced gut leakiness: a) miR-212 is highly expressed in intestine;b) EtOH increases miR-212 expression in ALD;c) EtOH disrupts intestinal tight junctions by affecting miR-212's target gene, ZO-1. Thus, we hypothesize that: EtOH-induced miR-212 expression causes the disruption of tight junctions by down regulating its target gene, ZO-1, through NFkB and iNOS signaling. This disruption causes gut leakiness that is associated with ALD. To test this hypothesis, two specific aims will be pursued. 1. Determine whether EtOH-induced upregulation of NFkB and iNOS signaling increase miR-212 expression in intestinal epithelial cells. We will use both pharmacological (specific inhibitors) &molecular (siRNA) to inhibit activation of NFkB or upregulation of iNOS to see if alcohol can still increase miR-212 and monolayer hyperpermeability. 2. Translate our in vitro observation to an in vivo model to establish the key role of miR-212 in EtOH-induced gut leakiness. To investigate whether miR-212 is needed for alcohol-induced gut leakiness in vivo, we will use LNA"""""""" microRNA Knockdown Probes to knockdown miR-212 expression in animal model of ALD. To determine whether iNOS mediate the EtOH-induced miR-212 expression and gut leakiness in vivo, we will feed EtOH to iNOS Knockout mice. Significance: The leaky gut plays an important role in ALD and other diseases, such as inflammatory bowel disease and colon cancer. Understanding the role of miRNA in regulation of the intestinal permeability could lead to novel preventive and therapeutic strategies for leaky gut in general and ALD in particular.

Public Health Relevance

Heavy drinking is a major health problem in the US, consuming 15% of total health costs. Heavy drinking- induced alcoholic liver disease has high morbidity and mortality with no satisfactory therapy. This proposal is designed to study the role of microRNAs in the mechanisms of alcohol-induced gut barrier dysfunction that is associated with alcoholic liver disease. Disturbance of intestinal barrier function is also associated with many human diseases such as Crohn's disease, ulcerative colitis, and colon cancer. Understanding the role of microRNA in the regulation of gut barrier function could lead to the development of novel therapeutic strategies.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AA018729-01A1
Application #
7991541
Study Section
Health Services Research Review Subcommittee (AA)
Program Officer
Murray, Gary
Project Start
2010-07-15
Project End
2012-04-30
Budget Start
2010-07-15
Budget End
2011-04-30
Support Year
1
Fiscal Year
2010
Total Cost
$225,000
Indirect Cost
Name
Rush University Medical Center
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
068610245
City
Chicago
State
IL
Country
United States
Zip Code
60612
Tang, Yueming; Zhang, Lijuan; Forsyth, Christopher B et al. (2015) The Role of miR-212 and iNOS in Alcohol-Induced Intestinal Barrier Dysfunction and Steatohepatitis. Alcohol Clin Exp Res 39:1632-41
Patel, Sheena; Behara, Rama; Swanson, Garth R et al. (2015) Alcohol and the Intestine. Biomolecules 5:2573-88
Tang, Yueming; Forsyth, Christopher B; Keshavarzian, Ali (2014) The role of miRNAs in alcohol-induced endotoxemia, dysfunction of mucosal immunity, and gut leakiness. Alcohol Clin Exp Res 38:2331-4
Forsyth, Christopher B; Voigt, Robin M; Shaikh, Maliha et al. (2013) Role for intestinal CYP2E1 in alcohol-induced circadian gene-mediated intestinal hyperpermeability. Am J Physiol Gastrointest Liver Physiol 305:G185-95
Tang, Yueming; Forsyth, Christopher B; Keshavarzian, Ali (2011) New molecular insights into inflammatory bowel disease-induced diarrhea. Expert Rev Gastroenterol Hepatol 5:615-25
Forsyth, Christopher B; Tang, Yueming; Shaikh, Maliha et al. (2011) Role of snail activation in alcohol-induced iNOS-mediated disruption of intestinal epithelial cell permeability. Alcohol Clin Exp Res 35:1635-43
Forsyth, Christopher B; Tang, Yueming; Shaikh, Maliha et al. (2010) Alcohol stimulates activation of Snail, epidermal growth factor receptor signaling, and biomarkers of epithelial-mesenchymal transition in colon and breast cancer cells. Alcohol Clin Exp Res 34:19-31
Miranda, Rajesh C; Pietrzykowski, Andrzej Z; Tang, Yueming et al. (2010) MicroRNAs: master regulators of ethanol abuse and toxicity? Alcohol Clin Exp Res 34:575-87