Screening of antioxidant components in Scutellariae Radix through mediating Nrf2-ARE signaling pathway based on estrogen receptor / 中国药理学通报

Aim: To establish ARE dual-luciferase reporter assay system and used it to identify the antioxidant substance of Scutellaria baicalensis Georgi. Methods: 293T cells were transiently co-transfected with ARE luciferase reporter plasmid PGL 4. 37 and sea kidney luciferase reporter plasmid PRL-TK. Three major active ingredients of Scutellaria baicalensis Georgi such as scutellarin, baicalein, baicalin and/or estrogen receptor (ER) specific inhibitor were added to Nrf2-ARE luciferase reporter assay system to detect whether they exerted antioxidant effect through the estrogen receptor affecting the Nrf2-ARE signaling pathway. Results: Baicalin (100 μmol · L-1) could obviously activate Nrf2-ARE pathway in 293T cells, and the induced expression was(1. 56 ±0. 01) times that of blank group (P < 0. 01). After pre-administration of ER specific inhibitor, the induced expression decreased to (1. 02 ±0. 23) times, and the antioxidant effect disappeared. After pre-administration of ER and Nrf2-ARE pathway specific inhibitor respectively, ROS in HaCaT cells injured by UVB significantly increased and and SOD was markedly down-regulated by baicalin. Conclusion Baicalin plays antioxidant activity through mediating Nrf2-ARE signaling pathway based on estrogen receptor.

Construction of dual luciferase reporter plasmid of HIPK3 3′UTR and verification its targeting miR-146 / 基础医学与临床

Objective To construct a dual luciferase reporter vector containing the 3′untranslated region(3′UTR) of HIPK3 gene and verify the relationship between HIPK3 and miR-146.Methods The binding sites of miR-146 and HIPK3 genes were predicted by miRDB database and DIANA TOOLS database. The 3′UTR region sequences of HIPK3 genes and its mutants were respectively inserted into the luciferase report plasmid psiCHECK-2 to construct a wild-type and a mutant recombinant dual luciferase report plasmid. The 293T cells were divided into 6 groups and transfected with 1) HIPK3-WT+NC negative control;2)HIPK3-WT+miR-146a mimics;3)HIPK3-WT+miR-146b mimics;4)HIPK3-MU+NC negative control; 5)HIPK3-MU+miR-146a mimics and 6)HIPK3-MU+ miR-146b mimics respectively. After 48 hours, the luciferase activity was detected.Results HIPK3-WT and HIPK3-MU re-combinant plasmid were successfully constructed. When HIPK3-WT recombinant plasmids and miR-146b mimics were transfected into 293T cells, the luciferase activity was decreased (P<0.05). Conclusions miR-146a does not have a target relationship with HIPK3 gene,whereas miR-146b can regulate the 3′UTR of HIPK3 gene.

Effect of -1 SNP on CCNH promoter activity / 基础医学与临床

Objective To study the effect of-1 site single nucleotide polymorphism(SNP) on CCNH gene promoter transcription activity.Methods PCR and site-directed mutagenesis technology were used to construct CCNH basic promoter and-1G mutate promoter.Dual-Luciferase Reporter assay system was used to detect the transcription activity of constructed promoter.Results In AD293 cells,the activity of-1G mutate type promoter was significantly lower than that of wild type-1T promoter(P

Role of transcription factors Sp1 and Sp3 in the expressional regulation of ezrin in esophageal carcinoma cells / 中国免疫学杂志

Objective:To identify the role of transcription factors Sp1 and Sp3 in the expressional regulation of ezrin in human esophageal carcinoma cells.Methods:Esophageal carcinoma EC109 cells were transfected with expressing vectors CMV-Sp1 or CMV-Sp3,and the effect of Sp1 and Sp3 over-expression on ezrin mRNA and protein expression was determined by real time RT-PCR and Western blot analysis.Furthermore,EC109 cells were cotransfected with the ezrin promoter-directed luciferase reporter vector and control vector pRL-TK along with transcription factor expression vector.The roles of Sp1 and Sp3 in ezrin promoter activation and whether this activation occurred through the Sp1 binding site,-75/-69,were analyzed by dual-luciferase reporter assay system.Results:Over-expression of transcription factors Sp1 and Sp3 significantly increased the expression of ezrin mRNA and protein and the ezrin promoter activity in EC109 cells.Sp1 and Sp3 enhanced the promoter activity through different binding sites and only Sp1 did that through the-75/-69 site.Conclusion:Sp1 and Sp3 can regulate ezrin expression in EC109 cells.