HCV NS2TP Gene Regulation Mechanism
|School||Dalian Medical University|
|Keywords||HCV NS2 NS2TP gene promoter CREB|
Objictive:NS2 trans-regulated protein (NS2TP) gene is one of the trans-regulated genes of the HCV NS2 in Suppression subtractive hybridization (SSH). In our studies, we validated that NS2 trans-regulates NS2TP gene from translational level and transcriptional level, identified the important sites of the NS2TP gene promoters, and demonstrated its transcription regulation mechanism.Methods:(1) To validate that HCV NS2 can trais-activate NS2TP gene. First, transfect HepG2 cells with the expression vector of pcDNA3.1(–)-NS2, and pcDNA3.1(–) as negative control. Extracting total protein and total RNA, we performed Western Blot experiment in translation level, and Real-time PCR experiment in transcription level, respectively. To analyze and study HCV NS2 can trans-activate NS2TP gene.(2) To analyze the activity of NS2TP gene promoter, and confirm the core area. Firstly, we successed in constracting expression vector system of dual-luciferase report gene, pGL4.10-NS2TP-promoter, and transfecting HepG2 cells by different parts of NS2TP gene promoter. On the basis of analysis on the promoter activity, we confirmed the activity core area of the NS2TP gene promoter, and analyzed that the NS2 is regulated with NS2TP gene promoter.(3) To validate that transcription regulation factor, CREB, acts with promoter core area. On bioinformatics technology, we analyzed the core area of promoter, identified the CREB binding site by EMSA and ChIP, and studied whether CREB involve in the transcription regulation of NS2TP gene by point mutation, overexpression CREB, and inhibition experiment.Results:(1) By Real-time PCR, we confIrmed HCV NS2 can up-regulate the transcriptional level of NS2TP gene. By Western Blot, we confirmed that HCV NS2 can promote the expression of NS2TP gene. (2) The minimal activate area of NS2TP gene was located the up stream 257 ribonucleotides of the transcriptional start site (–257-bp to +93-bp), and we also validated HCV NS2 can up-regulate the activity of NS2TP gene promoter.(3) On the basis of EMSA and ChIP, we confirmed that CREB is the important regulation proteion of NS2TP gene, as well as CREB involve in the regulation of NS2TP gene by point mutation, overexpression CREB, and inhibition experiment.Conclusion:(1) HCV NS2 trans-activate NS2TP gene.(2) The core area of NS2TP gene promoter is located in–257-bp to +93-bp.(3) Transcription regulation factor, CREB, involves in NS2TP gene transcriptional regulation.