**7.2 miR-212 expression in animal model of ALD**

To translate our in vitro observation to an in vivo model to establish the key role of miRNA-212 in EtOH-induced gut leakiness, we turned to use an animal model of alcohol-induced gut leakiness. This model has been established and validated in our previous studies. Recently, to determine whether gut leakiness and endotoxemia are one of the key co-factors for development of alcoholic steatohepatitis (ASH), we studied time courses for development of gut hyperpermeability, endotoxemia, and liver injury and showed that gut leakiness and endotoxemia occurred several weeks prior to development of ASH. These data support the notion that gut leakiness causes endotoxemia, which leads to alcoholic steatohepatitis and serious ALD.

In this animal model, rats were given daily EtOH (6 g/kg, by gastric gavage) for 10 weeks. The miRNAs expression levels in the intestine mucosa were assayed by miRNA microarray analysis (Miltenyi Biotec, Auburn CA). The data show that miR-212 expression in EtOH treated group was up regulated by 1.5 fold compared with control group (**Fig.4**).

To translate our in vitro observation to an in vivo model to establish the key role of miRNA-212 in EtOH-induced gut leakiness, we turned to use an animal model of alcohol-induced gut leakiness. This model has been established and validated in our previous studies. Recently, to determine whether gut leakiness and endotoxemia are one of the key co-factors for development of alcoholic steatohepatitis (ASH), we studied time courses for development of gut hyperpermeability, endotoxemia, and liver injury and showed that gut leakiness and endotoxemia occurred several weeks prior to development of ASH. These data support the notion that gut leakiness causes endotoxemia, which leads to alcoholic

In this animal model, rats were given daily EtOH (6 g/kg, by gastric gavage) for 10 weeks. The miRNAs expression levels in the intestine mucosa were assayed by miRNA microarray analysis (Miltenyi Biotec, Auburn CA). The data show that miR-212 expression in EtOH

treated group was up regulated by 1.5 fold compared with control group (**Fig.4**).

Fig. 3. Duble-log scatter plot

steatohepatitis and serious ALD.

**7.2 miR-212 expression in animal model of ALD** 

Fig. 4. miR-212 expression levels in intestinal mucosa were increased in rats of ALD model. The rats were fed with EtOH (6 g/kg, by gastric gavage) for 10 weeks. The miR-212 expression levels were assayed by miRNA microarray analysis
