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.