Abstract 1394: Endogenous 27nt microRNA inhibits eNOS Expression through DNA Methylation
Introns in eukaryotic genes have been recently recognized as important regulators in gene expression. Previously, we have reported that the numbers of the 27nt repeats in eNOS intron 4 were associated with cardiovascular risk; and demonstrated that 27nt repeats give rise to 27nt microRNA, which directly inhibits eNOS expression. In the present study, we investigated how the 27nt microRNA regulates eNOS expression. We examined the histone acetylation in the eNOS promoter and the upstream region of intron 4 in human aortic endothelial cells (HAECs) treated with 27nt microRNA using ChIP assay. We showed a significant deacetylation in histone H3 covering the eNOS promoter (−238 to −13bp, and −483 to −312bp) and the upstream region of the 27nt repeats (4932 to 5092bp). Using a direct sequencing method, we further evaluated the DNA methylation status. The CpGs within the region of 645 nucleotides upstream of the 27nt repeat (up to exon 3) were heavily methylated in HAECs treated with 27nt microRNA (treated: 73.4 ± 8.1% vs untreated: 8.2 ± 1.1%, p < 0.01). This methylation was associated with a reduced eNOS expression (eNOS mRNA in treated: 0 17 ± 0.03 vs untreated: 0.92 ± 0.24, p < 0.01). However, the inhibition was alleviated by the AzadC treatment - a DNA methylation inhibitor (0.89 ± 0.17). While conventionally microRNA suppresses gene expression by promoting mRNA degradation (perfect match at 3′UTR) or inhibiting translation (imperfect match), our data suggest a novel nuclear microRNA-based regulatory mechanism by modifying the status of target DNA methylation and histone acetylation, which affect the accessibility of the essential transcription factors for the eNOS gene, hence bioavailability of the vascular NO. Our study provides the first evidence that the intron-derived microRNA can effectively inhibit the expression of its own source gene by reducing transcription efficiency. This intron-based negative feedback self-regulatory model could be applicable to other genes that share the similar genomic structure as the repeat element in eNOS intron 4. Understanding this regulatory mechanism can lead to further development of tools to fine-tune the physiological eNOS expression that is critical for vascular integrity and disease. (NIH HL066054, AHA EIA).