猪NCOA3启动子甲基化对其在肌内和皮下脂肪组织差异表达的影响

[目的]以新淮猪肌内脂肪和皮下脂肪为试验材料,探讨核受体辅激活蛋白3(NCOA3)启动子甲基化对其在肌内与皮下脂肪组织中差异表达的影响。[方法]采用RT-PCR的方法检测NCOA3在肌内脂肪与皮下脂肪组织的表达水平。构建NCOA3启动子缺失报告载体,并采用双荧光报告基因系统分析启动子区域活性。预测NCOA3启动子区CpG岛,利用亚硫酸氢盐法(BSP)检测2种脂肪组织NCOA3启动子区CpG岛甲基化水平,并通过甲基转移酶处理,以检测甲基化对NCOA3基因启动子活性的影响。[结果]NCOA3 mRNA在肌内脂肪组织中表达水平极显著低于皮下脂肪组织(P<0.01),其中-150~-3 bp区域启动子活性最高,CpG岛位于-930~-699 bp,且其整体甲基化水平在肌内脂肪与皮下脂肪组织中差异不显著(P>0.05),分别为90.0%,83.5%,但-904位点CG甲基化水平差异显著(P<0.05)。经甲基转移酶处理pLUC-1057(包含CpG岛)载体荧光活性显著下降(P<0.05)。[结论]NCOA3基因在肌内脂肪与皮下脂肪组织存在着表达差异,-904位点CG甲基化水平对肌内与皮下脂肪组织表达差异存在影响。
[Objectives]We explore the effect of the nuclear coactivator 3(NCOA3)promoter methylation on its different expression patterns in intramuscular and subcutaneous adipose tissue in Xinhuai porcine. [Methods]The mRNA expression of NCOA3 in intramuscular and subcutaneous adipose tissue was determined by RT-PCR. Analysis of the activity of NCOA3 promoter of was identified by dual-luciferase reporter system. Methylation level was detected by sodium bisulfite genomic sequencing for the CpG island in NCOA3 promoter. Effects of methylation level on promoter activity were determined through in vitro methylation analysis. [Results]The mRNA expression of NCOA3 in intramuscular adipose tissue was significantly lower than in subcutaneous adipose tissue(P<0.01). NCOA3 core promoter and CpG island were located in -150 to -3 bp and -930 to -699 bp region respectively. The whole methylation level of CpG island showed no differences between intramuscular(90.0%)and subcutaneous(83.5%)adipose tissue. However,the methylation level of -904 CG site in intramuscular fat(55%)was higher than that in subcutaneous fat(20%)(P<0.05). Methyltransferase treatment of NCOA3 promoter region,which increased methylation level of CpG island,led to significant depression of gene acti-vities(P<0.05). [Conclusions]The expression of NCOA3 was significantly different in intramuscular and subcutaneous adipose tissue. Methylation level of -904 CG site might have effect on NCOA3 differential expression in intramuscular and subcutaneous adipose tissue