To Study The Regulation Of Expression Of GCLC Gene

ObjectiveReporter genes are commonly used to test the effect of promoter on the gene expression and theinteractions with the trans-factors, through measuring the expression of reporter gene to evaluatethe effects of regulatory sequences indirectly. Nowadays the dual luciferase enzyme reportanalysis system is the most used, of which,one is used to test promoter activity and the otheras an internal calibration. Our group has used this method for a long time to analyze the up-streamregulatory sequences. β-NF, belonging to falconoid, is a typical inducer of many antioxidantenzymes. when studying the and the two-phase drug, it is often used as a positive control. In ourstudy when we used this method to test the effect of β-NF on the expression of human GCLCgene, we found that our results were contrary to reports as discovered.This problem has troubledus for a long time, so we designed different experiments to observe the β-NF activity of theluciferase reporter gene.Method1. the dual luciferase enzyme report analysis system to screen external stimuli: the use of humanGCLC gene regulatory sequences driving of GCLC-PGL3-the enhancer-luciferase reported vector(PL45) transfected cells, adding different stimulating factors (different concentrations) tostimulate,the dual luciferase reporter gene assay systems analysis of GCLC gene expression;2.Western blot to detect endogenous GCLC protein expression using β-NF stimulation.3.he dual-luciferase reporter gene assay system to analysis β-NF on Luciferase gene expression:analysis of β-NF stimulated transfected in the expression of the Luciferase the eukaryoticexpression vector pRC/CMV2-luc which impact on the Luciferase gene expression; PL45weretransfected into cells, lysis of the cells with β-NF stimulation, analysis of β-NF Luciferase geneexpression.Result1.In different stimulis, β-NF strongly inhibit GCLC expression, this effect of β-NF was contraryto the repors; In A549cells,by adding two kinase inhibitor we found that the inhibition of β-NFhad nothing to do with MAPK or GSK-3β pathways.2.In different tissue-derived cells, the results showed that β-NF still had a strong inhibitory effecton the expression of human GCLC gene; Western Blot showed that β-NF can up-regulate theendogenous GCLC protein level; when transfected with non-β-NF reaction vector, the resultshowed that it also inhibit GCLC expression strongly; we collected and lysis the cells and then treated with β-NF, the results showed that β-NF had a strong inhibition, and it had nothing to dowith the expression.ConclusionLuciferase reporting gene system was used for the regulation of gene expression. In our study wefound that β-NF inhibit Luciferase genes direcly. So when we use this to analyze this system tostudy the two-phase detoxification enzymes we should consider the effects of drugs on reportinggenes and should use more methods so we can get a better results. ObjectiveReporter genes are commonly used to test the effect of promoter on the gene expression and theinteractions with the trans-factors, through measuring the expression of reporter gene to evaluatethe effects of regulatory sequences indirectly. Nowadays the dual luciferase enzyme reportanalysis system is the most used, of which,one is used to test promoter activity and the otheras an internal calibration. Our group has used this method for a long time to analyze the up-streamregulatory sequences. β-NF, belonging to falconoid, is a typical inducer of many antioxidantenzymes. when studying the and the two-phase drug, it is often used as a positive control. In ourstudy when we used this method to test the effect of β-NF on the expression of human GCLCgene, we found that our results were contrary to reports as discovered.This problem has troubledus for a long time, so we designed different experiments to observe the β-NF activity of theluciferase reporter gene.Method1.-850~-782bp range transcriptional regulation of experimental study: site-directed mutagenesis-810~-769bp range of the AP-2and NF-κB components and inspection of the GCLC geneexpression; examining different length deletions reported vectors on the GCLC gene expression.2.EMSA experiment to find the-850~-782bp range function of regulatory elements;3.DNA-Protein Pull-Down method to explore transcription factor-850~-782bp region.Result1.We successfully constructed site-mutagenesis of NF-κB and AP-2components reported vector,transfection results showed that NF-κB and AP-2component had the negative effects2.Transfection of deletion mutants of fragments of different lengths-838~-810bp was a negativeregulatory region,-810~-782bp was a positive regulatory region.3.EMSA and DNA-Protein Pull-Down showed that, of GCLC gene upstream regulatoryregion-838~-815bp and-810~-782bp were nucleoprotein combination, but had not yet found thepossible transcription factor. Conclusion-810~-769bp of NF-κB and AP-2components were negative regulatory elements;-850~-782bpregion still needs further exploration...