title: Regulation of endothelial nitric oxide synthase by small RNA.
abstract: Repeats (27-nt) in intron 4 have been shown to play a cis-acting role in endothelial nitric oxide synthase (eNOS) promoter activity. We hypothesize that the 27-nt repeats could be the source of small nuclear RNA specifically regulating eNOS expression. In this study, we used synthesized 27-nt RNA duplex and found that the eNOS gene transcriptional efficiency was reduced 63% (0.047 +/- 0.009 vs. 0.126 +/- 0.015, P < 0.01) by nuclear run-on assay. In endothelial cells transfected with the 27-nt small RNA duplex, we found that the eNOS mRNA and protein levels were decreased by >64% (P < 0.01). Conversely, a randomly selected 27-nt from luciferase gene had no effect on the eNOS expression. Furthermore, this eNOS silencing effect appeared to be reversible under the stimulation of vascular endothelial growth factor (10 ng/ml), which is known to up-regulate eNOS expression. Using in situ hybridization and Northern blotting, we observed the presence of endogenous eNOS intron 4-derived 27-nt small RNA, which was confined to the nucleus. In summary, we demonstrated that intron-based microRNAs in eNOS can induce significant gene specific transcriptional suppression, which could be an effective negative feedback regulator for gene expression.
authors.affiliation: Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Ming-Xiang
authors.initials: MX
authors.lastname: Zhang
authors.forename: Hesheng
authors.initials: H
authors.lastname: Ou
authors.forename: Ying H
authors.initials: YH
authors.lastname: Shen
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Jian
authors.initials: J
authors.lastname: Wang
authors.forename: Joseph
authors.initials: J
authors.lastname: Coselli
authors.forename: Xing Li
authors.initials: XL
authors.lastname: Wang
mesh_terms: D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007438:Introns; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology
chemical_list: D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; D052250:Nitric Oxide Synthase Type III