IDENTIFICATION AND ELUCIDATION OF MIR-214 AND MIR-29A AS POTENT REGULATORS OF LIVER FIBROSIS
Knabel, Matthew Kevin
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Liver fibrosis is the accumulation of extracellular matrix proteins in response to hepatocellular injury. Worldwide, liver fibrosis and cirrhosis accounts for nearly 160,000 deaths per year. It results from many different etiologies, including excessive alcohol consumption, viral infections and obesity. Our group has a vested interest in the molecular mechanisms that underlie this disease. Many patients on a solid organ transplant waiting list have excessive fibrosis, and those who receive a transplant often develop complications associated with recurrent fibrosis. One of the main objectives of our lab is to identify novel biomarkers of liver fibrosis, which will open avenues to understand novel mechanisms. Using this approach, we also test and develop therapeutic options to ameliorate liver fibrosis. This thesis focuses on identifying and understanding microRNA (miRNA) changes during fibrotic conditions. miRNAs are small, single-stranded RNA molecules that negatively regulate protein translation. In the following studies, we systematically examine changes in miRNA expression during liver fibrosis and identify two novel mechanisms that have cell-specific expression and function in the liver during liver fibrosis. In our first study, we describe a family of miRNAs that is upregulated during human and rodent liver fibrosis. This group of miRNAs, the miR-214/199 family, has five specific unique mature sequences that have been reported to regulate a variety of different targets. In the liver, expression of this family is focused predominantly in nonparenchymal cells. One member of this miRNA family, miR-214-5p, regulates expression of Twist1, a mesenchymal transcription factor that promotes expression of this family. Using in vitro techniques, we characterize this novel negative feedback loop in activated hepatic stellate cells. Inhibition of Twist1 is associated with downregulation of alpha-SMA, Col1a1 and CTGF, and reduces activation and migration of stellate cells and expression of the miR-214/199 family. Inhibiting miR-214-5p is associated with increased alpha-SMA, Col1a1 and stellate cell migration. Taken together, these findings suggest a novel negative feedback loop that regulates accumulation of fibrosis in the liver. In the next chapter, we report that miR-29a is downregulated significantly during liver fibrosis. miR-29a targets many extracellular matrix proteins, including collagen, elastin and fibrillin. Using an adeno-associated virus, we expressed miR-29a in hepatocytes of mice treated with carbon tetrachloride (CCl4). We report a dramatic therapeutic benefit when maintaining miR-29a expression in hepatocytes. Using an in vitro approach, we show that miR-29a is packaged into exosomes, and miR-29a is transferred from hepatocytes to stellate cells to regulate collagen expression. Finally, we create an inducible miR-29a transgenic mouse, and future directions the lab will pursue to further characterize these therapeutically beneficial observations. This work establishes roles of two different miRNA families that are dysregulated during liver fibrosis. Our efforts are great examples that point towards the need to consider miRNA expression on a cell-specific level. Characterizing other miRNAs would lead to identification of specific biomarkers, novel mechanisms and individual therapeutic options in nearly every pathogenic setting.