(DCP009) EFFECTS OF CIRCULAR RNA ZNF521 ON DIABETIC CARDIAC FIBROSIS

Background: Cardiac fibrosis resulting from diabetic cardiomyopathy (DCM) is a leading cause of heart failure in Canada. Fibrosis due to extracellular matrix (ECM) protein accumulation may occur through endothelial to mesenchymal transition (EndMT). Recent research has shown involvement of non-coding RNAs in DCM. CircularRNAs (circRNAs) are a class of non-coding RNAs that are covalently closed loops and have been implicated in various biological processes. Recent studies have suggested that circRNAs may play a role in diabetic cardiomyopathy. In addition, such effects may be mediated through miR-9, targeting ECM protein, we investigated circRNA ZNF521 (circZNF521) expression and its regulatory mechanism in cardiac fibrosis in diabetes through its interaction with miR-9.

METHODS AND RESULTS: We used endothelial cells (ECs) in vitro and a STZ induced diabetic mouse model to test our hypothesis. CircRNA microarray analysis identified circZNF521 expression in the diabetic mice hearts and was validated by qRT-PCR. miR-9 level (a target of circZNF521) and ECM proteins targeted by miR-9 were examined. Changes of EndMT markers were performed using qRT-PCR. circZNF521 siRNA was used to determine the interaction between circZNF521 and its target miR-9 and downstream protein in the ECs. Cardiac tissues from control and diabetic mice (B6 and endothelial specific miR-9 transgenic generated by us) were examined similarly.
The bioarray analysis showed high glucose (HG)-induced upregulation of circZNF521 and validated by qRT-PCR in ECs and mouse hearts. Simultaneously downregulation of miR-9 was also present in ECs and mouse hearts in diabetes. Furthermore mRNA expressions of EndMT markers, CD31 and VE-cadherin were decreased, α-SMA and FSP1 were increased in ECs after treatment with HG and in the hearts in diabetes. Such changes were prevented by circZNF521 silencing in the ECs. The relationship between miR-9 and circZNF521 was confirmed using circZNF521 siRNA transfection in ECs. Furthermore miR-9 antagomir co-transfection with circZNF521 siRNA partially recovered the expressions of ECM proteins in vitro. In the hearts of the diabetic animals, miR-9 overexpression was able to prevent the development of cardiac fibrosis.

Conclusion: These studies indicate a novel glucose-induced mechanism of increased ECM production, which is regulated by circZNF521 through miR-9. These findings may lead to potential RNA based prevention and treatment for DCM.