基于荧光素酶双报告基因系统体外验证长爪沙鼠Cip4基因受RXR及T4调控

In vitro verification of Cip4 gene regulation by RXR and T4 in Mongolian gerbils using luciferase double-reporter gene system

目的 探讨核转录因子TRB、RXR和激动剂T4对长爪沙鼠Cip4基因表达的调控作用。方法 合成长爪沙鼠Cip4基因启动子区序列,构建报告载体p GL3-Cip4;将转录因子TRB、RXR克隆到pc DNA3. 1表达载体上。将p GL3-Cip4,pc DNA3. 1-TRB,pc DNA3. 1-RXR和pRL-TK(参照质粒)载体共转染到HEK-293细胞,构建Cip4基因的荧光素酶双报告系统。观察质粒用量、TRB的激动剂甲状腺素T4等处理对转录活性的影响。结果 以长爪沙鼠Cip4启动子区为启动序列的双荧光素酶报告基因系统在(启动子+转录因子):pRL-TK=20∶1、总质粒量为2μg时转染效率最高,并具有最高的荧光素酶活性。检测结果显示,仅加入TRB不改变Cip4的转录水平(P>0. 05),仅加入RXR可以增加Cip4的转录水平(P<0. 05),RXR与T4共同作用可上调Cip4的转录水平(P<0. 001)。TRB与T4共同作用可下调Cip4活性(P<0. 05)。RXR、TRB、T4共存时,Cip4的转录水平显著下调(P<0. 01)。结论本研究证实了T4、TRB及RXR共调控长爪沙鼠Cip4基因的转录,模拟了配体激活型转录因子TRB/RXR活化目的基因Cip4的转录,同时证实了Cip4的转录活化与RXR信号通路密切相关,为揭示甲状腺素在人类NAFLD中的作用机制奠定了基础。本报告基因系统可用于配体激活型转录因子调控Cip4基因表达的机理分析及相关药物的筛选。

Objective To investigate the regulatory effect of nuclear transcription factors thyroxine receptor(TRB)and retinoic acid receptor X(RXR),as well as the agonist T4,on Cdc42-interacting protein 4(Cip4)gene expression.Methods The Cip4 gene promoter of Mongolian gerbils was cloned into the luciferase-reporter sequence of pGL3 basic to construct a new plasmid pGL3-Cip4.Coding sequences of transcription factors TRB and RXR were cloned into pcDNA3.1 eukaryotic expression vectors.pGL3-Cip4,pcDNA3.1-TRB,pcDNA3.1-RXR,and pRL-TK(reference plasmid)vectors were used to transfect HEK-293 cells to form a luciferase double-reporter system for the Cip4 gene.Transfection efficiency was optimized by adjusting the ratio of these plasmids.The agonist T4 was used to analyze transcriptional activity.Results Transfection efficiency and luciferase activity were the highest when a total amount of 2μg plasmid and a ratio of 20∶1 between the(promoter+transcription factor)and pRL-TK.RXR could significantly increase Cip4 gene transcription of(P<0.05),whereas TRB did not alter Cip4 transcription(P>0.05).Thyroxine T4 could enhance RXR upregulation(P<0.001),while a combination of T4 and TRB downregulated Cip4 transcription(P<0.05).Levels of Cip4 transcription were significantly downregulated in the presence of T4,RXR,and TRB(P<0.01).Conclusions T4,TRB,and RXR had coregulatory effects on Cip4 gene transcription in Mongolian gerbils;moreover,they simulated the process of ligand T4 agonizing TRB/RXR to activate Cip4 gene transcription.These result indicating that Cip4 transcription was significantly affected by the RXR signaling pathway lay the foundation for revealing thyroxine’s mechanism of action in human nonalcoholic fatty liver disease,as well as the study of ligand-agonizing nuclear transcription factor regulation of Cip4 gene expression and related drug screening.