【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。更多还原

【Abstract】 Objective To study the effect on the DNA methylation level of MS-1cell by inhibiting the expression of Clock gene,and explore the mechanism underlying Clock gene regulation of cell proliferation.Methods We knockeddown the expression of Clockgene by RNA interference,detected the DNA methylation level by Methylated DNA Quantification Kit,and the expression of Mecp2andβ-Catenin by Real-time PCR and Western blot.Results Knock-down the expression of Clockgene causes reduced DNA methylation level of MS-1cell,and the expression of Mecp2andβ-Catenin.The DNA methylation levels of Clockgene Koncked-down group and Neagtive Control group(NC)were 1.067±0.034%and 1.983±0.154%,p<0.05,respectively.The Real-time PCR results display the expression of Mecp2andβ-Catenin in CK group were 0.98824±0.12243 and 1.00000±0.09547,compare with the NC group 1.80979±0.18520and1.62584±0.17167,respectively.The relative protein levels of MECP2/β-TUBLLIN andβ-CATENIN/β-TUBLLIN were0.289546±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-1cell could be affected by Clock gene.Clock gene may regulate cell proliferation by inhibiting the expression of Mecp2andβ-Catenin.更多还原