THE EFFECT OF MIR-29 AND TGFB ON FIBROSIS
Creamer, Tyler Jones
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Fibrosis is defined as excess accumulation of extra cellular matrix (ECM) that impedes normal function of the parenchyma and is the cause of up to 45% of deaths in the developed world. With no favorable therapeutic strategy, development of new therapeutic interventions is necessary. Transforming growth factor beta (TGF-ß), a potent pro-fibrotic agonist, promotes deposition of ECM in part through upregulation of collagens, the major structural components of fibrotic scars. Loss of miR-29, a potent anti-fibrotic miRNA family that is regulated by TGF-ß and that targets multiple collagen species, is often associated with fibrosis. In previous work, our laboratory showed that maintenance of miR-29a via self-complementary adeno-associated virus (scAAV) transduced to the liver was sufficient to ameliorate liver fibrosis in a carbon tetrachloride (CCl4)-induced liver fibrosis murine model. Conversely, targeting of miR-29b to hepatocytes was unable to recapitulate the scAAV result with miR-29a. To systematically interrogate the efficacy of miR-29 as a therapeutic agent in different cell populations, we generated a tetracycline-regulated miR-29a and green fluorescent protein (GFP) reporter transgenic mouse to temporally and spatially control miR-29a expression. Using 2 different constitutive reverse tetracycline-controlled transactivators (rtTA), transgene expression can be induced in many tissues and cell types with doxycycline. Unexpectedly, the induction of miR-29a to more than 3-fold normal levels across the liver was not sufficient to prevent CCl4-induced liver fibrosis. Upon investigation of the spatial expression of the transgene, we saw exclusion of GFP from the Acta2+, collagen-producing cells. Additional evidence suggested that the miR-29 transgene is being epigenetically regulated in high collagen-producing cells, which would be the desired therapeutic target of this miR-29a-based therapy. Using high throughput RNA sequencing and a physical interaction pulldown assay, we were able to infer function of miR-29a in the different liver cell populations in vitro in the presence or absence of synthetic injury. We showed that miR-29a interacted with ECM components and epigenetic regulators and was able to normalize TGF-ß induction of paracrine signals as well as non-coding RNAs. Collectively, my data indicate that miR-29a is a potent therapeutic when expressed in specific cells during fibrotic injury by regulating more than collagens.