苯中毒患者中抑癌基因p16高甲基化及其表达下调

Hypermethylation and downregulation of tumor suppressor gene p16 in benzene poisoning

目的研究苯是否通过引起DNA甲基化使抑癌基因p16转录失活。方法应用病例-对照研究对11名苯中毒患者和8名与苯中毒患者年龄(±5岁)、性别匹配,并且同工种同工龄的接苯但无苯中毒的对照进行检测。分别用实时荧光定量PCR及焦磷酸测序检测基因表达及甲基化水平。结果 p16的表达水平在苯中毒组(0.53)低于对照组(2.06)(P=0.064)。p16启动子区CpG位点甲基化平均水平苯中毒组(12.4%)高于对照组(11.3%)(P>0.05)。甲基化与基因表达水平相关分析发现,p16 CpG位点平均甲基化水平与基因表达水平负相关(pearson相关系数r=-0.64,P>0.05),其中p16启动子区第4个CpG位点位于嗅神经元转录因子共有结合序列内,其甲基化水平与基因表达水平显著负相关(pearson相关系数r=-0.88,P<0.05)。结论在苯中毒患者中p16的表达水平显著降低,基因启动子区CpG岛的高甲基化可能与该基因的低表达有关。需要进一步扩大样本量深入研究苯相关疾病抑癌基因表达异常的分子机制。

Objective To investigate whether benzene negatively affects the expression of p16 through DNA methylation.Methods We carried out a case-control study in Chinese occupational benzene poisoning patients.Eleven cases of BP and 8 controls who were matched for age（± 5years）,sex,working duration and job title with BP were recruited.Expression level was examined by quantitative real-time PCR.Bisulfite-PCR pyrosequencing was used to quantitate the level of DNA methylation.Results The expression levels of p16 are down-regulated in BP patients compared to the control group（0.53 versus 2.06,P=0.064）.The average percentage of methylated cytosines of p16 was higher in BP group than in controls（12.4%,11.3%,respectively,P0.05）.p16 mRNA level decreased with increasing methylation（Pearson r=-0.64,P0.05）.The fourth CpG site in p16 promoter is located within the consensus binding sequence for olfactory neuron-specific transcription factor.A significant negative correlation between mRNA level and the fourth CpG site was exhibited（Pearson r=-0.88,P0.05）.Conclusion Our report demonstrated that mRNA expression of p16 is significantly downregulated in BP patients.Hypermethylation in promoter CpG islands is likely to contribute to the downregulation of p16.Further in-depth studies,utilizing large number of samples,are needed to fully understand the molecular mechanism involved in the tumor-suppressor gene inactivation in benzene-related diseases.