内质网应激条件下CHOP直接调节p53基因的转录来抑制结肠肿瘤生长的机制研究

CHOP(C/EBP homologous protein) plays an important role in regulating endoplasmic reticulum (ER) stress response, but the mechanisms of CHOP contributing to the induction of apoptosis and suppression of tumorigenesis still remain ambiguous. Recently,we have identified CHOP as a candidate transcriptional factor which might be invovlved in the regualtion of p53 transcription by yeast one-hybrid combined with bioinformatic approach.However, the detailed molecular basis particularly its biological significance of CHOP in regulating p53 transcription and suppressing the growth of colonic carcinoma under endoplasmic reticulum stress needs to be further investigated. To this end, we have designed the following experiments:..1. We have generated p53 gene promoter including the deleted mutants reporter plasmids to identify the possible binding site(s) of CHOP protein to the p53 gene promoter..2.We will assure the direct binding of the CHOP protein to the p53 gene promoter in vitro and in vivo by EMSA and chromatin-immunoprecipitation (CHIP)..3.We will determine the effect of overexpression or silencing of CHOP expression on the mRNA expression of p53 by Northernblot or Taqman-PCR..4.We will determine the expression of CHOP and p53 mRNA under the endoplasmic reticulum stress by treating the cells with tunicamycin or thapsigargin..5.We will study and compare the effect of silencing of CHOP or p53 on apoptosis and cell cycle distribution under ER stress both in cell and xenografted mouse model...Our study will help to dissect the direct role of CHOP in regulating p53 transcription in response to ER stress and identify novel upstream and/or downstream signaling pathways of p53 in mediating CHOP-dependent tumor suppression of colonic carcinomas .

CHOP是内质网应激（ER）通路中的重要信号分子，目前对其在ER诱导的细胞凋亡及肿瘤发生中的作用机制所知有限。最近我们通过酵母单杂交并结合生物信息学技术发现CHOP可能直接参与p53基因的转录调节，但其具体机制及生物学重要性不清楚。本项目拟通过构建p53基因启动子突变体报告基因来发现CHOP蛋白与p53基因启动子区域结合的可能位点；利用EMSA和染色质免疫沉淀技术来验证CHOP蛋白与p53基因启动子区域的直接结合。通过过度表达或抑制CHOP的表达来研究其对p53基因在转录水平的影响；研究内质网应激压力对CHOP表达及其对p53转录水平的影响。通过建立有效的动物模型并结合小动物生物荧光显像技术来研究在内质网应激条件下CHOP通过调节p53基因的转录来抑制结肠肿瘤生长的作用机制。重要的是通过发现ER通路中参与p53调控的上,下游调节因子可为结肠肿瘤治疗发现新靶标。

CHOP是内质网应激（ER）通路中的重要信号分子，其在ER诱导的细胞凋亡及肿瘤发生中的作用机制尚不十分清楚。我们前期通过酵母单杂交结合生物信息学技术发现CHOP可能参与p53基因的转录。在本项目中我们通过构建p53基因启动子报告基因突变体，发现了CHOP蛋白可与p53基因启动子区域结合的精确位点；同时利用凝胶电泳阻滞实验（EMSA）和染色质免疫沉淀实验（CHIP）验证了CHOP蛋白与p53基因启动子区域的直接结合。在项目中新增了利用生物芯片结合生物大分子互作系统（SPRi影像技术）建立检测转录因子-DNA结合的方法，发现CHOP蛋白可在体外与p53基因启动子区域结合。 同时我们研究了过度表达CHOP或p53基因对彼此基因转录的影响，发现了CHOP与p53基因的直接调控关系。我们检测了Tunicamycin (TM) 与Thapsigargin (TG) 诱导的内质网应激对CHOP、p53表达及细胞凋亡的影响。我们的研究结果阐明了CHOP基因与p53基因的相互调控关系，揭示了内质网应激条件下CHOP通过调节p53基因的转录来抑制结肠肿瘤生长的作用机制。重要的是通过研究发现CHOP参与调节p53介导的信号传导通路及相关上、下游调节及效应分子为肿瘤的预防和治疗提供了新的靶标和途径。