哺乳动物细胞POLK、POLH、POLI基因对甲基硝基亚硝基胍的应答反应及其启动子区转录因子结合部位的生物信息学分析

Expression response to methyl-nitro-nitrosoguanidine and the bioinformative analysis of binding sites of transcription factors of POLK, POLH, POLI gene in mammalian cells

目的:观察甲基硝基亚硝基胍(MNNG)对培养细胞DNA聚合酶κ、η、ι编码基因表达的影响并用生物信息学方法预测它们的启动子区和启动子区中的转录因子结合部位。方法:利用半定量RT-PCR观察POLK、POLH、POLI基因表达改变。利用在线生物信息学软件,确定它们的启动子区,然后,用转录因子预测软件并结合进化足迹法检出包括启动子区在内的3000bp碱基上游序列中的转录因子结合部位。结果:0.2μmol·L-1MNNG导致细胞POLH和POLI表达水平在处理后6-24h间逐渐上升,升高约1倍;而POLK则呈下降;通过转录因子预测软件分析,在POLK、H和I启动子区分别检出了384个、413个和689个推断的转录因子结合部位;通过进化足迹法分析有效地降低了假阳性率,推断的转录因子结合部位分别下降到158个、80个和40个。结论:低浓度MNNG导致POLH、POLI表达水平升高,而POLK表达水平降低;通过在线软件,我们成功地在POLK,POLH,POLI启动子区分别检出。158、80和40个推断的转录因子结合部位;进化足迹法分析能够有效降低预测软件的假阳性率。

AIM ： To investigate the effect of methyl - nitro - nitro soguanidine （ MNNG ） on POLK, POLH, POLl gene expression in FL cells, and find out the transcription factor binding sites in their promoter regions with the metheds of bioinformatios. METHODS： The expressive changes of POLK, POLH, POLl were observed with semiquantitative RT - PCR. The promoter regions of POLK, POLH, POLI were confirmed by online prediction programs. The putative transcription factor binding sites in these promoter regions were predicted using phylogenetic footprinting and Transfac position weight matrix. RESULTS： The expression of POLH, POLl was elevated about 1 fold and that of POLK decreased in 0. 2 μmol·L^-1 MNNG -treated cells, as compared with the untreated controls. 384,413 and 689 putative transcription factor binding sites were predicted in the promoter regions of POLK, POLH and POLl gene, respectively. After analyzed by phylogenetic footprinting, the number of putative sites in POLK promoter region was reduced to 158, and those of POLH and POLl were reduced to 80 and 40, respectively. CONCLUSION： After log concentration MNNG exposure, the expression of POLH and POLl was elevated, but that of POLK was decreased. Through online software, we have successfully predicted 158, 80 and 40 transcription factor binding sites in POLK, POLH and POLl promoter region respectively. The false positive rate were effectively reduced by the analysis of phylogenetic footprinting during predicting the transcription factor binding sites in their promoter regions.