**1. Introduction**

318 Biomarker

Tondini, C. & Hayes DF. (1989) Circulating tumor markers in breast cancer. *Hematol Oncol* 

Oxidative stress is responsible for a variety of degenerative processes in many human diseases, as either cause or effect. At present, some biomarkers of oxidative stress have been used to determine an individual's oxidative status in relation to disease conditions. However, their accuracy, sensitivity, or specificity needs to be improved. The development of novel biomarkers for oxidative stress is urgent.

Micro RNAs (miRNAs) are highly conserved regulatory molecules expressed in eukaryotic cells. They are short non-coding RNAs that regulate gene expression by binding to target mRNAs, which leads to reduced protein synthesis and sometimes decreased steady-state mRNA levels. Although hundreds of miRNAs have been identified, much less is known about their biological function. There are evidences that miRNAs affect pathways fundamental to metabolic control in higher organisms such as adipocyte and skeletal muscle differentiation. Also, some miRNAs are implicated in lipid, amino acid, and glucose homeostasis. Thus, miRNA abnormalities may contribute to common metabolic and systemic diseases where oxidative stress plays a key role in their pathogenesis. Indeed, there are evidences indicated that miRNAs are able to modulate the cellular response to oxidative stress both *in vitro* and *in vivo*. Therefore, miRNA may be novel biomarkers for oxidative stress.

We hypothesize that miRNAs may be biomarkers for oxidative stress because: (1) since miRNA are post-transcriptional gene regulators, they may be able to function as 'quick responders' to oxidative stress. For example, upon exposure to stress, miRNA may rapidly localize to P-bodies or stress granules to regulate key genes involved in the oxidative stress response. After the stress is mitigated, miRNA inhibition may be promptly abated, allowing

 \* Correponding Author

Using miRNA as Biomarkers to Evaluate the Alcohol-Induced Oxidative Stress 321

The most intuitive goals for a biomarker are to help diagnose symptomatic and presymptomatic disease and to provide surrogate endpoints to demonstrate clinical efficacy of new treatments(Ogino and Wang, 2007). The usefulness of the ideal biomarker of oxidative damage lies in its ability to provide early indication of disease and/or its

Oxidative Stress (ROS/RNS)

MDA AGEs

Protein carbonyl Nitrotyrosine

Fig.1 Biomarkers for oxidative stress

Fig. 1. Biomarkers for oxidative stress

relation to alcohol consumption.

Lipid Protein DNA Sugars miRNA

When investigating the status of alcohol-induced oxidative stress, it is unclear what the most appropriate biomarker is and how to measure them. At present, the biomarkers of oxidative stress/damage and the methods used to measure them are different among different study groups. Thus, it is difficult to compare the study findings in different groups to determine which one is the best biomarker to evaluate an individual's oxidative status in

In addition, the validity of many biomarkers remains to be established. The biomarkers that have been developed and currently been used to evaluate the oxidative stress have several shortcomings(Dalle-Donne et al., 2006), such as: (*a*) the limited specificity of the assay itself for the product of oxidative/nitrosative damage being measured; (*b*) the fact that the analyte being measured is not a specific product of a specific ROS/RNS; (*c*) the lack of sufficient sensitivity to detect concentrations of the product being measured in healthy individuals, thus not allowing the definition of a reference interval; (*d*) concentrations of the product being measured being influenced by external factors such as the lipid content of the diet; or

So far, several oxidative stress biomarkers have been used in clinics to assess patient's reaction to oxidative stress, their accuracy, sensitivity, or specificity need to be improved

(*e*) the assay being too invasive for in vivo investigations in humans.

8-OHdG 8-nitroguanine

1. make early diagnosis.

Known Biomarkers New Biomarkers ?

2. Monitor disease development. 3. Monitor treatment response.

miRNA profiles

progression (Fig. 1).

Isoprostanes

commencement of translation and expeditive restoration of cells back to their normal state; (2) since miRNAs regulate numerous targets, they have the capacity to powerfully and efficiently coordinate a stress response involving numerous genes; (3) owing to their small size and high stability, miRNAs may be less susceptible to certain types of stress, like genotoxic insults. Hence, their ability to modulate stress response would be less likely to be compromised under oxidative-stress. Given the recent development in the field of miRNA research, we predict that miRNA will be promised biomarkers for oxidative stress.

To test our hypothesis, we chose to study Alcoholic Liver Disease (ALD) as an oxidative stress model because: (1) it has high morbidity and mortality with no satisfactory therapy; (2) we showed that oxidative damage is the major mechanism for ALD; (3) we have established and validate that ALD model in rats is a good model for studies of oxidative stress. Our studies demonstrated that: (1). Nitric oxide-induced oxidative stress is required for alcohol-induced gut leakiness and liver damage in this model; (2) The miRNA expression profile was identified by miRNA microarray analysis. The miRNAs signatures were validated by TaqMan real time PCR assay. Our research results demonstrated that the differentially expressed miRNAs are the sensitive and specific biomarkers for alcoholinduced oxidative stress. (3) We showed that oats supplementation, a diet with strong antioxidative effect that is widely used in diets to prevent many diseases associated with oxidative damage, prevents ALD in rats by preventing alcohol-induced oxidative tissue damage.

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 damage in animal models. The application of miRNA as new biomarkers will lead to: a) identification susceptible individuals who are at risk for oxidative stress and would thus benefit from interventions that provide antioxidants; b) novel strategies to prevent and treat oxidative injury.
