**7.1 Relative miRNA expression in rat intestine of ALD model**

Fluorescence signals of the hybridized miRXploreTM Microarrays were detected using a laser scanner from Agilent (Agilent Technologies). In figure 2 a representative false-color image of the microarray experiment is shown as an example; Red color indicates that the Hy5 signal intensity is higher than the Hy3 signal intensity. Therefore, the corresponding gene is overexpressed in the Hy5-labeled sample. Green spots, however, indicate that the fluorescence intensity in the control sample is stronger than in the experimental sample. Yellow spots indicate that the signal intensities are equal for both samples. The signal intensities of each spot/miRNA that passed the quality filtering are shown in a double-

endotoxemia and alcoholic steatohepatitis. Furthermore, we showed that oats supplementation prevents loss of intestinal barrier integrity, endotoxemia and steatohepatitis(Keshavarzian et al., 2001; Tang et al., 2009a). However, the mechanism for

Numerous markers of oxidative stress and antioxidant status have been evaluated, but there has been little systematic effort to validate sensitive and specific biomarkers for oxidative

Development of miRNA as a new biomarker for alcohol-induced oxidative stress is limited by the lack of easy access to the tissues from patient populations. Therefore, the majority of discovery work will need to be carried out in the animal model of ALD. The advantage provided by animal models is the ability to control and define disease stages. We have to correlate these model diseases to the clinical status of actual patient populations. The process of biomarker discovery in animal models will be validated by clinical studies. As the technology develops to allow higher throughput screening of miRNA, these candidate biomarkers can be more easily tested and applied to larger

The *long term goal* of our laboratory is to design an effective therapeutic intervention to prevent and treat oxidant-induced disorders. Increasing our understanding of the mechanism of oxidant-induced gut leakiness should lead to identification of optimal targets for development of new preventive and therapeutic strategies for alcohol-induced, endotoxin mediated, tissue damage such as ALD. Our studies should thus lead to development of: **a)** miRNA as biomarkers for identifying susceptible individuals who are at risk for endotoxemia & ALD and would thus benefit from interventions that prevent gut leak; **b)** novel strategies to prevent ALD by preventing gut leakiness and endotoxemia in alcoholics; **c)** novel therapies to treat advanced alcoholic and non-alcoholic liver disease, because gut leakiness can perpetuate the hepatic necroinflammatory cascade (via feedback loops) and thereby contribute to the progression of liver injury. Since our model involves oxidative stress and iNOS, the results could have relevance to other pathological conditions where gut leakiness and oxidative stress play key roles like non-alcoholic liver disease,

Fluorescence signals of the hybridized miRXploreTM Microarrays were detected using a laser scanner from Agilent (Agilent Technologies). In figure 2 a representative false-color image of the microarray experiment is shown as an example; Red color indicates that the Hy5 signal intensity is higher than the Hy3 signal intensity. Therefore, the corresponding gene is overexpressed in the Hy5-labeled sample. Green spots, however, indicate that the fluorescence intensity in the control sample is stronger than in the experimental sample. Yellow spots indicate that the signal intensities are equal for both samples. The signal intensities of each spot/miRNA that passed the quality filtering are shown in a double-

the protective effects of oats is unclear.

damage in animal models.

patient populations.

**7. Results** 

**6. Opportunity and potential impact**

inflammatory bowel disease & food allergy to name a few.

**7.1 Relative miRNA expression in rat intestine of ALD model**

logarithmic scale (Fig.3), represented by a dot. X-axis: Hy3 signal intensity, y-axis: Hy5 signal intensity. Dashed diagonal lines define the areas of x-fold differential signal intensities. Approximately 30 miRNAs including miR-212, miR-7, miR-145, and miR-146a are expressed primarily in digestive tract tissues.

Fig. 2. Hy5/Hy3 false-color image after scanning of microarray

The successful development of effective antioxidant therapies remains a key goal, the attainment of which is required to elucidate the role played by accumulation of oxidized molecules in clinical picture of disease associated with oxidative stress. The use of miRNA as a biomarker provides a logical scientific basis for major intervention trials of antioxidants; such trials could in turn eventually validate or disprove the biomarker concept.

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**2009a; Tang et al., 2009c)** 

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**miR-212 relative expression**

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Using miRNA as Biomarkers to Evaluate the Alcohol-Induced Oxidative Stress 327

**Control EtOH treated**

Fig. 4. miR-212 expression levels in intestinal mucosa were increased in rats of ALD model.

**8. Our recent publications related to the role of miRNA in the alcohol-induced oxidative stress (Keshavarzian et al., 2009; Tang et al., 2008; Tang et al.,** 

Alcoholic liver disease (ALD) is commonly associated with intestinal barrier dysfunction. Alcohol-induced dysregulation of tight junction proteins such as zonula occluden 1 (ZO-1) in intestinal epithelial cells, plays an important role 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. We previously showed that miR-212 levels in colon biopsy samples in ALD patients were higher than in healthy controls; while ZO-1 protein levels were lower. Here, we studied the mechanisms for the involvement of miR-212 in alcohol-induced gut leakiness. We showed that miR-212 is highly expressed in intestinal tissues using a TaqMan microRNA real time PCR assay. Alcohol-induced miR-212 over-expression is accompanied by reductions in ZO-1 protein expression, disruption of tight junction protein (ZO-1), and increased permeability of Caco-2 cell monolayers. Alcohol-induced

The rats were fed with EtOH (6 g/kg, by gastric gavage) for 10 weeks. The miR-212

**8.1 Effect of alcohol on miR-212 expression in intestinal epithelial cells and its** 

expression levels were assayed by miRNA microarray analysis

**potential role in alcoholic liver disease (Tang et al., 2008)** 

Fig. 3. Duble-log scatter plot
