Abstract 2284: MicroRNA-15b, -16, -195, And -424, Which Have the Same Seed Sequence, Regulate ADP-rybosylation Factor-like 2 And Cellular ATP Levels in Neonatal Rat Cardiac Myocytes
MicroRNAs (miRNAs or miRs) are small non-coding RNAs that modulate mRNA stability and post-transcriptional translation, and may play an important role in cardiac pathophysiology. While it is well known that mitochondrion, a highly abundant subcellular organelle in cardiomyocytes, undergoes various changes in cardiac diseases, the role of miRNAs in the regulation of mitochondrial function is poorly understood. In this study, we individually overexpressed a series of miRNA, highly expressed in the heart, in neonatal rat cardiomyocytes using lentiviral vector. Then, we assessed cellular ATP levels to evaluate mitochondrial integrity. As a result, we discovered miR-15b to decrease cellular ATP levels without affecting cell viability. Moreover, we found that miR-16, -195, and -424 possess the same seed sequence, the most critical determinant of miRNA targeting, as miR-15b, and that miR-16, -195, and -424 also decreased cellular ATP levels in the same manner as miR-15b. Through a bioinformatics approach, we identified ADP-rybosylation factor-like 2 (Arl2) as a potential target of miR-15b, -16, -195, and -424. Arl2 associates with adenine nucleotide transporter 1, which plays a crucial role in oxidative phosphorylation. Overexpression of these miRNAs suppressed the activity of a luciferase reporter construct fused with the 3′UTR of Arl2. In addition, miR-15b overexpression decreased Arl2 mRNA and protein levels. The effects of Arl2 siRNA on cellular ATP levels were similar with those of miR-15b, and the artificial expression of Arl2 could restore the ATP levels reduced by miR-15b. Electron microscopic analysis demonstrated small, electron-densed, and deformed mitochondria in cardiomyocytes transduced with miR-15b or Arl2 siRNA. To assess the functional consequences of silencing endogenous miR-15b in vitro, we used a lentiviral vector in which 3′UTR with tandem sequences complementary to miR-15b is linked to the luciferase reporter gene. The loss-of miR-15b function by this decoy resulted in increased Arl2 protein and cellular ATP levels. The present findings suggest that miR-15b, -16, -195, and -424 are involved in the deterioration of mitochondrial function by targeting Arl2 in cardiomyocytes.