An intronic miRNA regulates expression of the human endothelial nitric oxide synthase gene and proliferation of endothelial cells by a mechanism related to the transcription factor SP-1.
OBJECTIVE
This study was to investigate the molecular mechanisms underlying the 27nt-miRNA-mediated regulation of expression of the endothelial nitric oxide synthase (eNOS) gene.


METHODS
Cell lines overexpressing 27nt-miRNA or its mutant were established by transfecting the miRNA expression vector into the endothelial cells. eNOS mRNA and protein expression were examined by RT-PCR and Western Blotting, respectively. Luciferase activity reporter system was used to study the target of 27nt-miRNA.


RESULTS
The results showed that overexpression of 27nt-miRNA significantly inhibited eNOS mRNA level and protein expression, and reduced the eNOS transcriptional efficiency. Such inhibitory effects of 27nt-miRNA were attenuated by the sequence mutations in 27nt-miRNA. Interestingly, the transcription factor SP-1 expression was reduced by 27nt-miRNA. Meanwhile, overxpression of SP-1 protein partially restored eNOS expression, and rescued the 27nt-miRNA-mediated reduction of endothelial cell proliferation. Moreover, certain sites in the SP-1 mRNA were found to be the direct target of 27nt-miRNA by a luciferase reporter system.


CONCLUSIONS
These results demonstrate that the 27nt-miRNA suppresses eNOS gene expression and SP-1 expression in vascular endothelial cells. The 27nt-miRNA directly target to SP-1 mRNA, thereby contributing to proliferation of endothelial cells.
Center of Biochemistry Research, University of South China, Hengyang, Hunan, PR China.
Limei
L
Yan
Min
M
Kang
Zhengqi
Z
Qin
Wenyu
W
Zhang
Yumei
Y
Li
Hesheng
H
Ou
D001011:Aorta / Q000166:cytology; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000201:enzymology* / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology; D004794:Enzyme Repression; D015870:Gene Expression; D006801:Humans; D007438:Introns; D035683:MicroRNAs / Q000235:genetics*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics*; D034622:RNA Interference; D016329:Sp1 Transcription Factor / Q000235:genetics* / Q000378:metabolism
D035683:MicroRNAs; D016329:Sp1 Transcription Factor; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III