摘要目的 研究尼古丁诱导口腔癌前病变细胞中E26转录因子1(E26 transformation specific 1,Ets1)与过氧化物还原酶1(peroxiredoxin 1,Prx1)的相互作用,探讨尼古丁在口腔癌前病变中作用的分子机制.方法 培养口腔癌前病变细胞株(dysplastic oral keratinocyte,DOK),实验分为尼古丁组、对照组、敲低组和敲低对照组,尼古丁组、敲低组和敲低对照组DOK细胞用1μmol/L尼古丁处理7d,对照组不作任何处理.分别采用蛋白质印迹法、免疫共沉淀(co-immunoprecipitation,Co-IP)和染色质免疫共沉淀(chromatin immunoprecipitation,ChIP)法检测尼古丁组和对照组DOK细胞中Prx1和Ets1的蛋白表达量、Prx1和Ets1蛋白结合量以及Ets1与过氧化物还原酶1基因(PRDX1)启动子区的结合情况.分别利用siRNA干扰和慢病毒感染技术敲低Ets1和Prx1,蛋白质印迹法检测敲低组和敲低对照组DOK细胞中Prx1和Ets1的蛋白表达变化.结果 尼古丁诱导Prx1和Ets1蛋白表达显著增加,尼古丁组Prx1和Ets1蛋白相对表达量分别为0.71±0.02和0.12±0.01,均显著高于对照组中二者的表达(Prx1和Ets1蛋白分别为0.53±0.06和0.01±0.01,P=0.009,P=0.000).Co-IP结果显示,Prx1能与Ets1形成蛋白复合体,尼古丁组Prx1和Ets1蛋白结合量高于对照组.ChIP检测发现,尼古丁可显著上调Ets1与PRDX1基因启动子区的结合,尼古丁组富集倍数(80.9±19.7)显著高于对照组(13.8±1.2)(P=0.004).在DOK细胞中,成功敲低了Ets1和Prx1蛋白的表达,敲低组Ets1和Prx1蛋白相对表达量分别为0.60±0.06和0.48±0.03,敲低对照组分别为0.83±0.08和0.80±0.06 (P=0.016,P=0.002).敲低核转录因子Ets1可显著抑制Prx1蛋白表达(P=0.002);反之,敲低Prx1亦可显著降低Ets1蛋白的表达(P=0.000).结论 在口腔癌前病变细胞中,Ets1可直接调控Prx1的表达,与Prx1蛋白存在相互作用;尼古丁可能通过放大Ets1与Prx1之间的正反馈调控信号,促进口腔癌前病变的发生和发展.

abstractsObjective To investigate the interaction between nuclear transcriptional factor E26 transformation specific 1 (Ets 1) and peroxiredoxin 1 (Prx1) in nicotine-induced oral precancerous lesion cells.Methods Human oral precancerous lesion dysplastic oral keratinocyte (DOK) cells were cultured and divided into nicotine group,control group,knockdown group and knockdown control group.The nicotine group,knockdown group and knockdown control group were treated with 1 μmol/L nicotine for 7 days while the control group was untreated.Western blotting,co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (CHIP) were performed to detect Prx1 and Ets1 protein expression,Prx1 and Ets1 protein interaction,combined activity of Ets1 with PRDX1 gene promoter region in nicotine group and control group DOK cells.In nicotine group,DOK cells were transfected with siRNA or lentivirus to knockdown Ets1 and Prx1 expression.Prx1 and Ets1 protein expression was examined by Western blotting.Results Nicotine increased the expression of Prx1 and Ets1 protein in DOK cells.The relative expression of Prx1 and Ets 1 was 0.71±0.02,0.12±0.01 in nicotine group and 0.53±0.06,0.01±0.01 in control group (P=0.009,P=0.000).Co-IP showed that Prx1 could form protein complex with Ets1.The expression of Prx1 and Ets1 complex protein was increased in nicotine group.ChIP revealed that nicotine upregulated the combination of transcriptional factor Ets1 with PRDX1 gene promoter region,and the enrichment fold was 80.9± 19.7 in nicotine group and 13.8± 1.2 in control group (P=0.004).Ets1 and Prx1 protein expression was knocked down.The relative expression of Ets 1 and Prx1 was 0.60±0.06,0.48±0.03 in knockdown group and 0.83±0.08,0.80±0.06 in knockdown control group (P=0.016,P=0.002).Ets1 knockdown suppressed the expression of Prx1 (P=0.002).Conversely,Prx1 knockdown also inhibited the expression of Ets1 significantly (P=0.000).Conclusions In oral precancerous lesion cells,Ets1 directly regulates Prx1 expression and nicotine might promote the development of oral precancerous lesion by magnifying the positive feedback signal pathway between Ets 1 and Prx 1.