Alpha Omega Alpha Honor Medical Society

2014 Research Abstract

Regulation of PAI1 by miRNA-19a/b and Its Role in Pathogenesis of Pulmonary Arterial Hypertension

Investigator: Jason Huang, University of Illinois College of Medicine

Mentors: J. Usha Raj, MD, and Guofei Zhou, PhD

Rationale: Pulmonary arterial hypertension (PAH) is a debilitating and incurable disease. It is characterized by pulmonary vasoconstriction and vascular remodeling, which cause irreversible increases in pulmonary vascular resistance and arterial pressure, eventually resulting in right ventricular hypertrophy and failure. Studies suggest PAI1 expression in pulmonary arterial smooth muscle cells (PASMC) contributes to the pathogenesis of PAH. This study investigates PAI1’s regulation in PAMSCs; from the numerous forms of protein regulation, miRNA were determined to be strong candidates. Using data from our previous experiments and miRNA target prediction software, we determined miRNA- 19a and miRNA-19b (miR-19a and miR-19b, respectively) to be strong candidates for the elucidation of PAI1 regulation.

Methods: To investigate miR-19a/b regulation of PAI1, we transfected PASMC (Passage 7, 60mm dishes, 80-90% confluence) with 100nmol miR-19a or miR-b mimics or inhibitors using 15μL Lipofectamine 2000 transfection reagent. 48 hours after transfection, the cells were harvested. To determine the miRNA effect on PAI1 mRNA abundance, qRT-PCR was performed on mRNA extracted from the PASMCs. To determine the miRNA effect on PAI1 protein levels, Western Blots were done on the PASMC cell lysate. To determine whether miR-19a/b target PAI1’s 3’-UTR region, we constructed a luciferase reporter plasmid containing a copy of the PAI1 3’-UTR and cotransfected PASMCs with 100nmol miR-19a or miR-19b mimics or inhibitors and 2μg of the plasmid. After 48 hours, the cells were harvested and luciferase reporter assays were done on the cell lysate.

Results: Luciferase assays showed miR-19a/b mimics reduced luciferase reporter expression to approximately 0.5 of control levels (P<0.01). Interestingly, only miR-19a inhibitors induced reporter expression, while miR-19b inhibitors yielded no significant change. QRT-PCR data showed miR-19a/b mimics decreased PAI1 mRNA levels to approximately 0.5 of control levels (P<0.01); miR-19a inhibitors induced expression, while miR-19b yield a smaller induction of mRNA induction. At the protein level, miR- 19a/b mimics decreased PAI1 protein (P<0.05); consistently, miR-19a/b inhibitors increased protein levels (P<0.01).

Conclusion: The data suggest miR-19a and miR-19b inhibit PAI1 protein expression by targeting the 3’-UTR of PAI1 mRNA. Mimic miRNA data indicate miR-19a/b target PAI1 mRNA. Inhibitor data further implies miR-19a may play a larger role in the regulation of PAI1, because while inhibition of miR-19a was enough to greatly induce PAI1 expression, inhibition of miR-19b did not induce the same levels of expression, suggesting miR-19b’s inhibitory role is supplemented by other factors, such as miR-19a. These results suggest miR-19a/b may be regulating factors of PAI in PASMCs.

Last modified: 8/14/2015

Updated on August 14, 2015.

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