新型shRNA慢病毒载体抑制TGIF表达及对TGF-β通路的调节

Supression of TGIF by shRNA mediated in novel inducible lentiviral vector modulates TGF-β signaling

关键词:  可诱导慢病毒shRNA载体  TGIF  TGF-β信号通路  Tet-on系统

Key words:  inducible lentiviral vector  TGIF  TGF-βsignaling  Tet-on system

摘要:

目的 构建针对同源域蛋白TGIF（5′-TG-3′interacting factor）可诱导表达shRNA的慢病毒载体,鉴定其在大肠癌细胞系DLD1中对TGIF的基因沉默效率,进一步研究其对TGF-β信号通路下游基因的影响。方法 设计并合成针对TGIF的RNA干扰序列,克隆到可被强力霉素诱导的新型Tet-on慢病毒载体,将慢病毒颗粒感染大肠癌细胞系DLD1细胞。培养并分离细胞克隆后,使用强力霉素（doxcycline）进行诱导,用Real-Time PCR和Western印迹法检测TGIF和PAI-1的表达情况。结果 构建了针对TGIF可诱导shRNA的真核表达病毒载体并获得相应的病毒,病毒可以高效感染大肠癌细胞系DLD1。在强力霉素诱导下,TGIF在DLD1细胞系的表达得到有效抑制,TGF-β下游基因PAI-1和p15表达明显升高。结论 本研究成功构建了可诱导表达shRNA慢病毒载体,其在强力霉素诱导下可以有效抑制TGIF在大肠癌细胞系内的表达;TGIF可以抑制TGF-β信号传导通路。

Abstract:

Objective To construct inducible shRNA in lentiviral vector against homodomain protein TGIF gene and to investigate its gene silencing effect.Methods Three pairs of target sequences were synthesized and cloned into novel pTRIPZ Tet-on lentiviral vector which was induced by doxcycline. The expression vector was transfected into 293T cell,and the lentivirus was harvested from 293 T cells. Virus was used to infect DLD1 cells.The expression of TGIF,PAI-1 and p15 was detected by real-time PCR and/or Western blot.Results The shRNA of TGIF inducible lentiviral vector was succes-sfully constructed.The lentivirus was also obtained and mediated by 293T,which were highly efficient to infect colon carcinoma DLD1 cells.The expression of TGIF was supressed under doxcycline induction of shRNA while the expression of TGF-βdownstream genes PAI-1 and pl5 increased.Conclusion The inducible lentiviral vector containing shRNA supresses TGIF expression effectively under doxcycline induction.TGIF can inhibit TGF-βsignaling target genes.