DNA甲基化抑制剂DAC处理对鸡Wnt10a基因表达及其启动子甲基化的影响  

The influence of DNA methylation inhibitor 5-Aza-2’-deoxycytidine treatment on Gallus Wnt10a gene expression and the promoter region methylation

作　　者：吴挺 陈绪靖 陈世豪[1] 胡序明[1] 崔恒宓[1,2,3] WU Ting;CHEN Xujing;CHEN Shihao;HU Xuming;CUI Hengmi(Institute of Epigenetics and Epigenomics/College of Animal Science and Technolog,Yangzhou University,Yangzhou 225009,China;Joint International Research Laboratory of Agricultural&Agri-Product Safety,Ministry of Education,Yangzhou University,Yangzhou 225009,China;Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses,Yangzhou University,Yangzhou 225009,China)

机构地区：[1]扬州大学动物科学与技术学院/表观遗传学及表观基因组学研究所,江苏扬州225009 [2]扬州大学教育部农业与农产品安全国际合作联合实验室,江苏扬州225009 [3]扬州大学江苏高校动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《扬州大学学报（农业与生命科学版）》2021年第1期1-6,共6页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金面上项目(91540117、81773013);江苏省高校优势学科建设工程项目(PAPD)。

摘　　要：Wnt10a基因在细胞增殖、分化及迁移中发挥着重要作用,参与调控机体生长发育的诸多生物学过程,但对鸡Wnt10a基因表达的表观遗传调控机制尚不清楚。为探究DNA甲基化对Wnt10a基因表达的影响,以DMSO处理为对照组,5-Aza-2’-deoxycytidine (DAC)处理为试验组,分别用0.25、0.5、1.0、2.5、5.0μmol·L^(-1)浓度的DAC处理鸡胚成纤维细胞DF-1,通过RT-qPCR分析比较Wnt10a基因在DAC和DMSO处理后的表达水平;同时通过NCBI数据获取Wnt10a启动子区序列,设计亚硫酸氢盐测序(BSP)引物,5.0μmol·L^(-1) DAC处理鸡胚成纤维细胞DF-1 48 h,焦磷酸测序分析鸡Wnt10a基因上游部分CpG位点的甲基化水平。结果表明:相对于DMSO处理,DAC处理Wnt10a基因表达量极显著上调,且上调幅度随DAC处理浓度的升高而升高;同时Wnt10a基因上游CpG位点甲基化水平显著下降。综合而言,DNA甲基化抑制剂DAC可显著降低鸡Wnt10a基因起始位点上游序列部分CpG位点的甲基化水平,并激活Wnt10a基因的表达。The Wnt10a gene plays an important role in cell proliferation, differentiation, migration and the multiple biological processes involved in regulating the body’s growth and development. However, epigenetic regulation mechanism of the Wnt10a gene is unclear yet. This study aimed at investigating whether DNA methylation of the Wnt10a gene affects the gene expression. Chicken embryo fibroblast DF-1 cells were treated with 0.25, 0.5, 1.0, 2.5 and 5.0 μmol·L^(-1) of 5-Aza-2’-deoxycytidine(DAC), respectively and then RT-qPCR was used for Wnt10a expression detection. Based on NCBI database, sequences of Wnt10a promoter region(about 2 000 bp) were obtained and primers for bisulfite PCR sequencing were designed. Chicken embryo fibroblast DF-1 cells were treated for 48 hours, genomic DNA was extracted and DNA methylation was analyzed by bisulfite PCR pyrosequencing. Results showed that compared to the DMSO treatment group(control), the treatment of DAC significantly increased Wnt10a expression(P<0.01), and the gene expression increased with increasing concentrations of DAC.In contrast,DNA methylation of Wnt10 apromoter decreased significantly.In general,DNA methylation inhibitor DAC can activate the Wnt10 agene expression by decreasing DNA methylation of Wnt10 apromoter.

关 键 词：鸡DF-1细胞 DAC Wnt10a基因 基因表达量 甲基化 亚硫酸氢盐测序 

分 类 号：S831.2[农业科学—畜牧学]