鼠口腔黏膜癌变过程中生物钟基因Per1与细胞周期基因昼夜节律的表达

Circadian rhythm variation of the clock genes Per1 and cell cycle related genes in different stages of carcinogenesis of buccal mucosa in animal model

目的 探讨生物钟基因Perl和细胞周期基因p53、细胞周期蛋白（Cyclin）D1、B1、周期蛋白依赖性激酶（cyclin-dependent kinases,CDK）在口腔颊黏膜癌变不同阶段的昼夜节律表达变化及与癌变发生的关系.方法 90只金黄地鼠置于光照和黑暗交替环境中饲养,用二甲基苯并蒽涂抹颊黏膜建立金黄地鼠颊黏膜鳞状细胞癌模型,分别在涂抹二甲基苯并蒽前、6周和14周后的24 h开灯后时间（hour after lights onset,HALO）的4、8、12、16、20和24 HALO的6个时间点处死动物,每个时间点5只,分别获取正常颊黏膜、癌前病变和鳞状细胞癌3个癌变阶段各昼夜6个时间的组织.常规切片在HE染色下观察组织癌变情况;用实时定量荧光PC R（real time PCR,RT-PCR）检测各时间点组织中Perl、p53、CyclinD1及B1、CDK1 mRNA的表达;行余弦分析,以中值、振幅和峰值位相时为指标分析各基因的表达在癌变不同阶段的昼夜节律变化特征.结果 Perl、p53、CDK1和CyclinD1 mRNA分别在癌变3个阶段中的表达均具有昼夜节律性（P＜0.05）;CyclinB1 mRNA仅在正常颊黏膜和鳞状细胞癌阶段中的表达具有昼夜节律性（P＜0.05）,而在癌前病变阶段表现为昼夜节律紊乱（P＞0.05）;Per1和p53mRNA表达的中值随着鳞状细胞癌的发展而显著降低（P＜0.05）,CyclinD1、B1、CDKlmRNA表达的中值随着鳞状细胞癌的发展而显著上升（P＜0.05）;Per1、p53和CyclinD1 mRNA表达的振幅随着鳞状细胞癌的发展显著降低（P＜0.05）,CDK1mRNA的振幅随着鳞状细胞癌的发展显著增加（P＜0.05）,而CyclinB1 mRNA的振幅无显著性改变（P＞0.05）;在癌前病变阶段Per1和CDK1 mRNA峰值出现时间（即峰值位相时）较正常阶段明显提前,而p53和CyclinD1 mRNA峰值表现为明显滞后;在鳞状细胞癌阶段CDK1和CyclinB1 mRNA的峰值位相时较正常阶段明显滞后,而Per1、p53和CyclinD1 mRNA无明显变化.结论 随着鳞状细胞癌的发生和发展,生物钟基因Per1和细胞周期基因p53、CyclinB1、D1、CDK1、Cyclin表达的昼夜节律特征明显改变.

Objective To investigate the expression and circadian rhythm variation of biological clock gene Per1 and cell cycle genes p53,CyclinD1,cyclin-dependent kinases （CDK1）,CyclinB1 in different stages of carcinogenesis in buccal mucosa and its relationship with the development of buccal mucosa carcinoma.Methods Ninety golden hamsters were housed under 12 hours light-12 hours dark cycles,and the model of buccal squamous cell carcinoma was established by using the dimethylbenzanthracene（DMBA） to smear the golden hamster buccal mucosa.Before the DMBA was used and after DMBA was used 6 weeks and 14 weeks respectively,the golden hamsters were sacrificed at 6 different time points （5 rats per time point） within 24 hour,including 4,8,12,16,20 and 24 hour after lights onset（HALO）,and the normal buccal mucosa,precancerous lesions and cancer tissue were obtained,respectively.HE stained sections were prepared to observe the canceration of each tissue.Real time RT-PCR was used to detect the mRNA expression of Per1,p53,CyclinD1,CDK1 and CyclinB1,and a cosine analysis method was applied to determine the circadian rhythm variation of Per1,p53,CyclinD1,CDK1 and CyclinB1 mRNA expression,which were characterized by median,amplitude and acrophase.Results The expression of Per1,p53,CDK1 and CyclinD1 mRNA in 6 different time points within 24 hours in the tissues of three different stages of carcinogenesis had circadian rhythm,respectively.However,the CyclinB 1 mRNA was expressed with circadian rhythm just in normal and cancer tissue （P〈0.05）,while in precancerous lesions the circadian rhythm was in disorder （P〉0.05）.As the development of carcinoma,the median of Per 1 and p53 mRNA expression were significantly decreased （P〈0.05）,yet the median of CDK1,CyclinB1 and CyclinD1 mRNA expression were significantly increased （P〈0.05）.The amplitude of Per1,p53 and CyclinD1 mRNA expression was significantly decreased as the development of carcinoma （P〈0.05）,however the amplitude of CDK1 mRNA expression was significantly increased （P〈0.05）.In addition,there was no significant difference in the amplitude of CyclinB1 mRNA expression.Tbe time that the peak expression value of Per1 and CDK1 mRNA appeared （Acrophase） in precancerous lesions was remarkably earlier than that in normal tissues,but the acrophase of p53 and CyclinD1 mRNA expression was remarkably delayed.Moreover,the acrophase of CDK1 and CyclinB1 mRNA expression in cancer tissues was obviously earlier than that in normal tissues,yet there was no significant variation in acrophase of Per1,p53,CyclinD1 mRNA expression between normal tissues and cancer tissues.Conclusions The circadian rhythm of clock gene Per1 and cell cycle genes p53,CyclinD1,CDK1,CyclinB1 expression remarkably varied with the occurrence and development of carcinoma.Further research into the interaction between circadian and cell cycle of two cycle activity and relationship with the carcinogenesis may provide new ideas and methods of individual treatment and the mechanism of carcinogenesis.