Construction of pGL3-TNF-α3′UTR luciferase reporter gene and tanshinone compounds screening
10.3969/j.issn.1001-1978.2015.01.017
- VernacularTitle:TNF-α3′-UTR双荧光素酶报告基因系统的构建及丹参酮类药物筛选
- Author:
Zhonghong WEI
;
Zhijie ZHU
;
Yuping LIU
;
Zhaoguo LIU
;
Xiaobo SHENG
;
Siliang WANG
;
Li TAO
;
Pinting ZHU
;
Wenxing CHEN
;
Aiyun WANG
;
Yin LU
- Publication Type:Journal Article
- Keywords:
TNF-α,3′UTR;
dual luciferase reporter;
post-transcriptional regulation;
cryptotanshinone;
com-pounds screening
- From:
Chinese Pharmacological Bulletin
2015;(1):77-81
- CountryChina
- Language:Chinese
-
Abstract:
Aim To screen the potential inhibitors of post-transcriptional activity of pro-inflammatory media-tor TNF-α from the lipophilic constituents in Chinese Medicine Salvia miltiorrhiza Bunge ( Danshen) , we es-tablished dual luciferase reporter gene system pGL3-TNF-α3′UTR ( 3′untranslated region ) co-transfected with Renilla control gene. Methods Complementary DNA ( cDNA) template was obtained from human um-bilical vein endothelial cells ( HUVECs ) . The full length DNA of TNF-α 3′-UTR was amplified through PCR, and then connected the luciferase reporter vector pGL3-control after enzyme digestion. pGL3-TNF-α 3′UTR constructs were co-transfected with pSVRenilla into the mononuclear macrophages RAW264. 7 cells. The relative activity of reporter genes was measured by dual luciferase reporter ( DLR ) assay system after the stimulus of lipopolysaccharide ( LPS ) in presence or absence of tanshinones compounds. Results The pGL3-TNF-α3′UTR luciferase reporter gene was suc-cessfully constructed. The cloning DNA fragment and sequence were both consistent with the GENBANK da-tabase. LPS significantly induced the relative reporter activityof RAW264 . 7 cells transfected with pGL3-TNF-α 3′UTR. Among four tanshinones compounds, we found only cryptotanshinone could significantly de-crease LPS-induced relative reporter activity. Conclu-sion The pGL3-TNF-α 3′UTR construct combined with DLR assay system was successfully established, which can be used to discover the agents such as cryp-totanshinone that regulate the post-transcription of TNF-α in treatment of inflammatory and malignant dis-eases.
