钟基因Per2和肿瘤相关钟控基因在金黄地鼠口腔颊黏膜癌变不同阶段的昼夜节律改变

Circadian variation of clock gene Per2 and cancer-related clock-controlled genes in buccal mucosa carcinoma of golden hamster at different cancer stages

目的探讨钟基因Per2和钟控基因血管内皮生长因子(VEGF)、Ki67、c-Myc和P53在颊黏膜癌变不同阶段的昼夜节律变化规律以及与癌变发生发展的关系。方法 90只叙利亚金黄地鼠置于12 h光照和12 h黑暗交替环境中饲养,用二甲基苯并蒽(DMBA)涂抹颊黏膜建立金黄地鼠颊癌模型,分别在DMBA涂抹前,涂抹6周和14周后的24 h内的6个不同时间点处死动物,获取正常颊黏膜、癌前病变和癌症3个不同阶段昼夜6个不同时间的组织。经病理学检查确认后,采用实时荧光定量聚合酶链反应检测各时间点Per2、VEGF、Ki67、c-Myc和P53 m RNA的相对表达量,并行余弦分析,以中值、振幅和峰值位相时为指标反映各基因表达的昼夜节律特征。结果 Per2、VEGF、P53和c-Myc m RNA在癌变3个阶段的表达均具有昼夜节律性(P<0.05),而Ki67 m RNA仅在正常黏膜和癌前病变阶段的表达具有昼夜节律性(P<0.05)。Per2和P53 m RNA表达的中值随着癌症的发展而降低(P<0.05),VEGF、c-Myc和Ki67 m RNA表达的中值随着癌症的发展而上升(P<0.05);P53 m RNA的振幅随着癌症的发展而降低(P<0.05),Per2、VEGF、Ki67和c-Myc m RNA的振幅在癌前病变和癌症阶段均高于正常组(P<0.05);在癌前病变阶段,Per2、VEGF和c-Myc m RNA的峰值位相时较正常组提前,而Ki67和P53 m RNA的峰值位相时较正常组滞后。结论随着癌症的发生和发展,钟基因Per2和肿瘤相关钟控基因VEGF、Ki67、c-Myc、P53表达的昼夜节律特征发生明显改变。

Objective This study investigates the circadian variation rules of the clock gene Per2 and clock-controlled genes of vascular endothelial growth factor （VEGF）, Ki67, c-Myc, and P53 in different stages of carcinogenesis in buccal mucosa carcinoma and their roles in the development of buccal mucosa carcinoma. Methods Ninety Syrian golden hamsters were housed under 12 h lighV12 h dark cycles. Dimethylbenzanthracene （DMBA） was used to establish the carcinoma model by smearing the golden hamster buccal mucosa. Before DMBA painting and after 6 and 14 weeks, the hamsters were sacrificed at six time points within a period of 24 h （i.e., 4, 8, 12, 16, 20, and 24 h after light onset）, and the normal buccal mucosa, precancerous lesions, and cancer tissues were simultaneously obtained. Hematoxylin and eosin stained sections were prepared to observe the canceration of each tissue. Real time polymerase chain reaction was used to detect the mRNA expression of Per2, VEGF, Ki67, c-Myc, and P53. Cosine analysis was employed to determine the circadian-rhythm variations of Per2, VEGF, Ki67, c-Myc, and P53 mRNA expression in terms of median, amplitude, and acrophase. Results The expression of Per2, VEGF, P53, and c-Myc mRNA in three different stages appeared with circadian rhythms （P〈0.05）, whereas the Ki67 mRNA was expressed with circadian rhythm only in normal and precancerous lesion stages （P〈0.05）. The midline-estimating statistic of rhythms （MESORs） of Per2 and P53 mRNA were significantly down-regulated with the development of cancer （P〈0.05）, whereas the MESORs of VEGF, c-Myc, and Ki67 mRNA were up-regulated （P〈0.05）. The amplitude of P53 mRNA significantly decreased with the development of cancer （P〈0.05）. Moreover, compared with the normal group, the amplitudes of Per2, VEGF, Ki67, and c-Myc mRNA significantly increased in precancerous lesions and cancer tissue （P〈0.05）. In precancerous stage, the acrophases of Per2, VEGF, and c-Myc mRNA were earlier than that in the normal group, whereas that of Ki67 and P53 mRNA were delayed. Conclusion The circadian-rhythm characteristics of the clock gene Per2 and clock-controlled gene expression of VEGF, Ki67, c-Myc, and P53 mRNA have changed with the occurrence and development of carcinoma.