抑制MS-1细胞Clock基因表达引起DNA甲基化水平降低  

Knock-down the expression of Clock gene causes reduced DNA methylation level of MS-1 cell

作　　者：李世平[1] 成姝婷 王正荣[2] 刘延友[2] 汪宇辉[2] 江舟[2] 

机构地区：[1]四川大学华西第二医院儿科.出生缺陷与相关妇儿疾病教育部重点实验室,四川成都610041 [2]四川大学华西基础医学与法医学院时间生物学卫生部重点实验室,四川成都610041

出　　处：《西部医学》2017年第8期1054-1057,1062,共5页Medical Journal of West China

基　　金：国家自然科学基金(31371108;31500935)

摘　　要：目的探讨Clock基因对MS-1细胞甲基化水平的影响及Clock基因参与调控细胞增殖的机制。方法采用siRNA干扰MS-1细胞Clock基因表达,检测细胞DNA甲基化水平变化,以及甲基化结合蛋白MECP2,影响细胞生长和增殖的Wnt/β-Catenin信号通路β-Catenin基因的表达情况。结果与对照组相比,抑制Clock基因表达后,MS-1细胞甲基化水平显著降低,且Mecp2和β-Catenin基因的表达均明显减少。抑制Clock基因组(CK)和阴性对照组(NC)甲基化DNA占总DNA比率分别为:(1.067±0.034)%和(1.983±0.154)%。Real-time PCR结果表明,CK组β-Catenin基因相对表达量为1.00000±0.09547,NC组为1.62584±0.17167;CK组Mecp2为0.98824±0.12243,NC组为1.80979±0.18520。Western blot结果显示,CK组β-CATENIN/β-TUBLLIN的相对IOD值为1.093368±0.214433,NC组为3.305374±0.260016;CK组MECP2/β-TUBLLIN的相对IOD值为0.289546±0.104738,NC组为0.649544±0.140909。结论 Clock基因能够影响细胞甲基化水平,并且可能通过调控甲基化结合蛋白MECP2表达和Wnt/β-Catenin信号通路影响细胞增殖。Objective To study the effect on the DNA methylation level of MS-1 cell by inhibiting the expression of Clock gene, and explore the mechanism underlying Clock gene regulation of cell proliferation. Methods We knocked- down the expression of Clock gene by RNA interference, detected the DNA methylation level by Methylated DNA Quan- tification Kit, and the expression of Mecp2 and β-Catenin by Real-time PCR and Western blot. Results Knock-down the expression of Clock gene causes reduced DNA methylation level of MS-1 cell, and the expression of Mecp2 and β-Catenin. The DNA methylation levels of Clock gene Koncked down group and Neagtive Control group（NC） were 1. 067 ± 0. 034 and 1. 983%. 154% ,p〈0.05, respectively. The Real time PCR results display the expression of Mecp2 and β-Catenin in CK group were 0. 98824±0. 12243 and 1. 00000±0. 09547, compare with the NC group 1. 80979±0. 18520 and 1. 62584±0. 17167, respectively. The relative protein levels of MECP2/β-TUBLLIN and β-CATENIN/β-TUBLLIN were 0.289546±0.104738 and 1. 093368±0.214433 in CK group, compare with 0.649544±0.140909 and 3. 305374±0. 260016 in the NC group, respectively. Conclusion The DNA methylation level of MS-1 cell could be affected by Clock gene. Clock gene may regulate cell proliferation by inhibiting the expression of Mecp2 and β-Catenin.

关 键 词：DNA甲基化 CLOCK基因 MECP2 WNT/Β-CATENIN信号通路 

分 类 号：R722[医药卫生—儿科]