MicroRNA-1 inhibits cardiomyocyte proliferation in mouse neonatal hearts by repressing CCND1 expression.

Abstract

The functions of microRNA-1 (miR-1) in cardiac hypertrophy, and cardiomyocyte differentiation have been investigated. However, the mechanism on how miR-1 could repress cardiomyocyte proliferation has not been fully elucidated. We address this issue by investigating whether miR-1 affected the proliferation of neonatal cardiomyocyte and identify some of the genes targeted by miR-1. miR-1 was over-expressed in neonatal cardiomyocytes and the effect on cell cycle and growth were analyzed by flow cytometry and Brdu-incorporation assay. Relevant vectors carrying the luciferase reporter were constructed for validation of miR-1 binding to its matching sites on the 3'-untranslated region of the predicated target mRNAs. Cardiomyocytes were co-transfected with the vectors and miR-1 mimics, then luciferase reporter assay was performed. Lastly, we examined the expression of target genes in cardiomyocytes after transfection with miR-1 mimics, as well as their normal expression pattern in 2- and 13-day-old mice hearts. We have demonstrated that miR-1 was the most significantly upregulated miRNA in 13-day-old mouse hearts compared with 2-day-old hearts. We also showed that miR-1 could repress cardiomyocyte G1/S phase transition, proliferation and viability. IGF1 and CCND1 were identified as candidate target genes regulated by miR-1. In addition, overexpression of miR-1 could suppress the expression of these two genes at the mRNA level. It could also correspondingly inhibit CCND1 expression at the protein level but not for IGF1. Our results suggest that miR-1 plays an important role in inhibiting cardiomyocyte proliferation in the developing neonatal mouse heart by directly suppressing the cell-cycle regulator, CCND1.