Differential regulation of miRNA and mRNA expression in the myocardium of Nrf2 knockout mice

Nuclear Factor Erythroid-derived 2-like 2 (Nrf2) senses oxidative environments and/or stress and initiates a cytoprotective response through transcriptional activation of antioxidant and detoxification genes. Several preclinical studies suggest that Nrf2 combats oxidative stress underlying a variety of pathologies. Despite Nrf2 deficits linked to functional abnormalities in many organ systems, the transcriptional network resulting from Nrf2 deficiency in the heart has remained elusive. Moreover, cross-talk between microRNAs (miRNAs) and cardiac Nrf2 signaling is unknown. Here, we utilized next generation RNA sequencing (RNAseq) to unbiasedly profile basal mRNA and miRNA expression in Nrf2 knockout (Nrf2−/−) hearts. RNAseq of mRNA revealed 152 differentially expressed genes (DEGs) in the Nrf2−/−myocardium, of which 129 were downregulated. Grouping of DEGs based on biological function and real-time qPCR validation indicated that DEGs were enriched for; mitochondrial genome and bioenergetics, oxidoreductase capacity, cardiac development, and chaperone activity. Interestingly, RNAseq analysis uncovered 27 significantly altered miRNAs, of which 11 were upregulated and 16 were downregulated in Nrf2−/− hearts. Expression changes were validated for 12 miRNAs using specific primer assays in real-time and revealed a significant decrease in miR-10b-5p, miR-674-3p, miR-3535, and miR-378c while miR-30b-5p, miR-208a-5p, miR-350-3p, and miR-582-5p, and miR-1249-3p levels were increased. High throughput data were integrated using prediction algorithms, and these in silico analyses discovered potential recognition elements within 39 repressed mRNAs which matched the seed sequence for 4 upregulated miRNAs; miR-30b-5p, miR-208a-5p, miR-350-3p, and miR-582-5p. These high-throughput data reveal transcriptome-wide effects of myocardial Nrf2 deficiency. Further, our results suggest that Nrf2 may directly or indirectly regulate a sub-set of cardiac miRNAs in the basal setting. This comprehensive analysis is the first evidence to demonstrate a plausible regulatory cross-talk among cardiac miRNAs and the Nrf2 transcriptional network, and provides valuable candidates to examine in future mechanistic and preclinical studies.