HBx及HBs调节RhoC启动子机制的研究

Regulation of RhoC Promoter by HBx and HBs

目的探讨HBx（hepatitis B virus X protein）及HBs（hepatitis B virus S protein）对RhoC启动子的作用机制，分析可能相互作用的启动子调控元件。方法构建RhoC启动子截短突变克隆，双荧光素酶报告分析系统（the dual-luciferase reporter assay system）检测RhoC启动子相对荧光素酶活性，通过分析启动子相对活性变化进而筛寻到起调控作用的启动子区段。过表达Ets-1转录因子，检测RhoC启动子相对荧光素酶活性。结果①成功构建了5个RhoC启动子截短变异克隆。②分别在HepG2细胞和HepG2.2．15细胞中验证了RhoC截短变异启动子活性，确定了起关键作用的启动子区段（-491bp～-320bp、-124bp～-58bp）。③HepG2细胞中分别过表达HBx及HBs，发现启动子区段-491bp～-320bp及-187bp～-124bp可以被HBx及HBs调控。④文献检索结合软件分析这些启动子区段序列，找到了高度相关的转录因子Ets-1、NF-κB、Sp1等。⑤荧光素酶检测和Real-time PCR初步验证了转录因子Ets-1对RhoC的调控作用。结论HBx及HBs可能通过Ets-1等转录因子调控RhoC启动子进而上调RhoC启动子活性。

Objective To investigate the possible promoter regulatory elements in 5＇flanking sequence of RhoC promoter. Methods Five different 5＇-deletion luciferase reporter constructs of RhoC gene promoter were generated by using the 5＇-deletion assay. Their relative luciferase activity was determined by the dual-luciferase reporter assay system. The regulatory segments of the promoter were identified by detecting the luciferase activity of the promoter. After Est-1 was over-expressed, the activity of RhoC gene promoter was detected by the dual-luciferase reporter assay system. Results （ 1 ） Five different 5＇-deletion luciferase reporter constructs of RhoC gene promoter were success- fully established; （2） The regions -491 bp - -320 bp and -124 bp - -58 bp might play an im- portant role in the activity of RhoC gene promoter; （3） The fragments -491bp -320 bp and -187 bp - - 124 bp could be regulated by over-expressing HBx and HBs in HepG2 cells; （4） Multiple putative binding sites for the transcription factors Ets-1, NF-KB, Spl etc, in these promoter frag- ments were identified; （5） The luciferase assay and real-time PCR demonstrated the regulation of the transcription factor Ets-1 to RhoC. Conclusion regulated by HBx and HBs, and the transcription volved in this process.