Abstract 9561: Loss of Full Length Pumilio 1 Abrogates Gene P27 Silencing-Mediated Cell Proliferation and Secures Mirna-221 Mimics Therapeutics in the Heart

Abstract

Introduction: MiR-221 mimics have been demonstrated to be cardioprotective and anti-fibrotic in the treatment of myocardium infarction and heart failure. However, miR-221 is a known oncomiR as the upregulation of miR-221 and the downregulation of its target p27 are associated with tumor progression and poor prognosis. Therefore the treatment of miR-221 mimics raise safety concerns. Hypothesis: miR-221 fails to trigger P27 mediated neoplastic cellular proliferation due to loss of full length pumilio-1 (FL PUM-1) expression in vivo . Methods: H9c2, Hela and primary cardiac fibroblasts were used to study the regulations of miR-221 mimics on P27 by RT-qPCR and Western blot, cell proliferation by cell number, CCk-8 and Ki67 staining. The interaction with PUM1 was assessed by siRNA transfection, p27 3’UTR cloning and luciferase reporter assay. In vivo 2 days after miR-221 mimic (1mg/kg and 4mg/kg) i.v. injection in the rats, the liver, heart, kidney and muscle were collected to measure p27 and PUM1 by RT-qPCR and WB. Pum1 cDNA was cloned using primers flanking its start and stop codons, and inserted into pcDNA3.1 vector. 10 colonies from each tissue were sequenced to verify mRNA splicing variants. PUM1 cleavage were verified by the incubations of cathepsin K (CatK) inhibitor odanacatib (ODN). Results: in vitro miR-221 mimics significantly downregulated the expression of P27 and increased cell proliferation. FL PUM1 (140kDa) and isoforms of 105 and 90 kDa were detected. The transfections of siRNAs to knockdown PUM1 but not PMU2 abolished miR-221-mediated effects and direct binding of miR-221 on p27 3’UTR. In vivo following two dosages miR-221 mimic treatments, the expressions of miR-221 increased in the heart, liver, kidney and muscle for 6.8 and 17.3 fold, 33.4 and 506.5 fold, 2.2 and 3.5 fold, and no significant increases respectively. P27 was not affected. FL-PUM1 was not detected in all tissues, but only isoforms. 105 kDa was generated through alternative RNA slicing and 90 kDa by CatK cleavage. Conclusions: This is the first study to address the safety concerns with respect to the possible pro-proliferative effects of miR-221 mimic therapeutics in cardiovascular applications. It provides essential insights of the context-dependent nature of miRNA functionality.