Abstract 169: miR-21-5p Regulation of Lipid Content and Peroxidation in H9C2 Cells

Abstract

Cardiovascular pathologies are the leading single cause of death in chronic kidney disease (CKD) patients. We have found that the 5/6 nephrectomy model of CKD leads to an upregulation of miR-21-5p in the left ventricle 7 weeks after surgery, targeting peroxisome proliferator-activated receptor alpha (PPARα). PPARα is a regulator of fatty acid uptake and metabolism. In our model we find that suppression of miR-21-5p alters the expression of numerous genes involved with fatty acid oxidation and glycolysis, presumably through its regulatory action on PPARα and/or additional targets. We also find that 5/6Nx rats exhibit dyslipidemia and increased left ventricular lipid content at this time. In this study we evaluated the potential for knockdown or overexpression of miR-21-5p to regulate lipid content and peroxidation in H9C2 cells. Cells were transfected with anti-miR-21-5p (40nM), pre-miR-21-5p (20nM) or appropriate scrambled oligonucleotide controls. After 24 hours medium was changed and half of the cells from each transfection group were treated with lipid (0.66 mM oleic acid and 0.33 mM palmitic acid) for 48 hours (n=6/treatment group for each set of experiments). Lipid content, measured by AdipoRed assay (Lonza) was significantly increased with lipid treatment (nearly two-fold). Overexpression of miR-21-5p significantly attenuated this increase (228.0 ± 9.7 vs. 198.2 ± 8.9% of untreated control), while suppression of miR-21-5p augmented lipid content (235.8 ± 11.2 vs. 328.1 ± 12.3% of untreated control). These results were supported by imaging of Oil Red O stained cells. We found that the abundance of malondialdehyde (MDA), a product of lipid peroxidation, was significantly increased in response to lipid treatment. Overexpression of miR-21-5p reduced MDA content in untreated and lipid treated cells, suggesting that miR-21-5p reduces oxidative stress. Suppression of miR-21-5p had no effect on MDA levels. These results indicate that overexpression of miR-21-5p attenuates both lipid content and lipid peroxidation in H9C2 cells. Ongoing studies aimed at evaluation of alterations in fatty acid oxidation and oxidative stress will further aid in determining the functional impact of miR-21-5p on associated pathways in cardiac tissue.