小鼠子宫内膜基质细胞中HOXA10调控DNM1L表达机制的研究  

The regulation mechanism of HOXA10 on DNM1L expression in mouse endometrial stromal cells

作　　者：肖成[1] 胡进[1] 纪红军[1] 牛亚茹 焦建桥 戴玉健 徐银学[1] 

机构地区：[1]南京农业大学动物科技学院,江苏南京210095

出　　处：《南京农业大学学报》2016年第6期1023-1029,共7页Journal of Nanjing Agricultural University

摘　　要：[目的]本文旨在研究转录因子同源框A10基因(HOXA10)结合动力蛋白基因(DNM1L)启动子区域绑定位点,并调控DNM1L基因的表达机制。[方法]通过转录因子网站(TFSITESCAN)预测DNM1L启动子区域存在HOXA10的结合位点。分离怀孕1~7 d的小鼠子宫内膜,采用RT-q PCR检测其HOXA10和DNM1L mRNA表达量的变化规律。结合HOXA10过表达和siRNA干扰试验,分析小鼠子宫内膜基质细胞中HOXA10 mRNA表达量的变化如何影响DNM1L表达。构建DNM1L野生型和绑定位点突变型启动子区域(-1 749^-2 033 bp)荧光素酶报告载体,分别与HOXA10过表达载体和siRNA共转染到小鼠3T3细胞,检测DNM1L启动子区荧光素酶活性值变化。[结果]网站(TFSITESCAN)预测DNM1L在转录起始位点上游-1 767 bp的位置存在HOXA10转录结合位点。HOXA10 mRNA表达量在怀孕4 d小鼠的子宫内膜中达到峰值,DNM1L mRNA表达量则在怀孕3 d时达到峰值。HOXA10过表达和siRNA干扰试验结果表明:基质细胞中HOXA10表达量升高或降低能相对应降低或提高DNM1L表达水平(P<0.01)。双荧光素酶活性试验结果表明:HOXA10过表达载体和siRNA分别与野生型DNM1L报告载体共转染细胞,前者荧光活性值极显著下降,后者荧光活性值极显著上升(P<0.01)。突变型DNM1L报告载体对荧光活性值无明显影响(P>0.05)。[结论]HOXA10可结合于DNM1L启动子区域,并调控DNM1L基因表达。[Objectives]This study was designed to verify that dynaminl-like gene （DNMIL）promoter region has binding sites of homeobox A10 gene（HOXA10）and investigate the mechanism for HOXA10 regulating DNMIL expression. [ Methods] We predicted that DNM1L promoter region has binding sites of HOXA10 by the transcription factors forecast wehsite（ TFSITESCAN）. We separated 1 to 7 days of female pregnant mice uterus endometrial, real-time quantitative PCR was used to detect the changes of HOXA10 and DNMIL mRNA expression in pregnant mice uterus endometrial. We combined HOXA10 overexpression and siRNA interference experiments to detect the influence of HOXA10 mRNA expression on DNMIL mRNA expression in mouse endometrial stromal cells. We constructed DNMIL wild-type and mutant binding sites luciferase reporter vectors in the promoter region（ -1 749 to -2 033 bp）. Then they were co-transfected into mouse 3T3 cells with HOXA10 overexpression vectors and siRNA respectively to detect DNM1L promoter region luciferase enzyme activity value. [ Results ] Website （TFSITESCAN）forecasted that there was a transcription binding sites of HOXA10 in DNMIL transcription start site - 1 767 bp position. HOXA10 mRNA expression reached peak at the fourth day of pregnancy in mice uterus endometrial. However,DNM1L mRNA expression reached peak on the third day of pregnancy. The experiments of HOXA10 overexpression and siRNA interference showed the increasing of HOXA10 mRNA expression significantly reduced DNM1L mRNA expression, while the decreasing of HOXA10 mRNA expression significantly increased DNM1L mRNA expression levels in stromal cells（P〈0.01 ）. Dual luciferase activity results showed that HOXA10 overexpression vectors and wild-type DNM1L luciferase reporter vectors were co-transfected into the cells, and then led to a significant decline of fluorescence activity value. However, co-transfecting of HOXA10 siRNA and the wild-type DNMIL luciferase reporter vectors significantly increased fluorescence activity value （P〈0.

关 键 词：同源框A10基因(HOXA10) 动力蛋白基因(DNM1L) 小鼠子宫内膜基质细胞 

分 类 号：S852.23[农业科学—基础兽医学]