Effect of NF-κB binding element deletion on transcriptional regulation of NOX1
10.3969/j.issn.1000-4718.2014.10.001
- VernacularTitle:NF-κB 结合元件缺失对 NOX1启动子转录活性的影响
- Author:
Weijing WU
;
Li LI
;
Caiyun XU
;
Wenjie HUANG
- Publication Type:Journal Article
- Keywords:
NADPH oxidase 1;
NF-kappa B;
Acute lung injury;
Promoter;
Transcription factor
- From:
Chinese Journal of Pathophysiology
2014;(10):1729-1734
- CountryChina
- Language:Chinese
-
Abstract:
AIM:To investigate the effect of NF-κB binding element deletion on transcriptional regulation of (NOX1).METHODS:pGL3-Basic vector inserted with the NOX1 proximal promoter, and the same vector inserted with the NOX1 proximal promoter in the absence of the positive NF-κB binding element, were constructed.After cloning, diges-tion and purification, NOX1 proximal promoter (≈1 415 bp) was inserted into the multicloning site of the pGL3-Basic vec-tor and then sequenced ( pGL3-NOX1-1415) .NF-κB binding elements in the NOX1 promoters were predicted by Alibaba 2.1 software.The positive element was deleted by overlapping PCR.The deletion mutant was inserted into the pGL3 vector in the same way (pGL3-NOX1-1327).The plasmids were transfected into A549 cells, and then the cells were stimulated with TNF-α.The luciferase activity was monitored on MD SpectraMax M5 enzyme-labeled instrument.RESULTS:The se-quences of pGL3-NOX1-1415 and pGL3-NOX1-1327 were identified to be correct.Compared with control group, the lucif-erase activity was significantly higher in the cells transfected with pGL3-NOX1-1327 (P<0.05), but it was significantly lower than that in the cells transfected with pGL3-NOX1-1415 (P<0.05).CONCLUSION: NF-κB plays an essential role in the transcriptional regulation of NOX1 in TNF-α-induced A549 cells.Activated NF-κB binds to specific elements in NOX1 promoter regions to control the transcription.
