Detection of Expressional Changes Induced by Intrauterine Growth Restriction in the Developing Rat Mammary Gland via Exploratory Pathways Analysis

BackgroundIntrauterine growth restriction (IUGR) is thought to lead to fetal programming that in turn contributes to developmental changes of many organs postnatally. There is evidence that IUGR is a risk factor for the development of metabolic and cardiovascular disease later in life. A higher incidence of breast cancer was also observed after IUGR. This could be due to changes in mammary gland developmental pathways. We sought to characterise IUGR-induced alterations of the complex pathways of mammary development at the level of the transcriptome in a rat model of IUGR, using pathways analysis bioinformatics.Methodology/Principal FindingsWe analysed the mammary glands of Wistar rats with IUGR induced by maternal low protein (LP) diet at the beginning (d21) and the end (d28) of pubertal ductal morphogenesis. Mammary glands of the LP group were smaller in size at d28, however did not show morphologic changes. We identified multiple differentially expressed genes in the mammary gland using Agilent SurePrint arrays at d21 and d28. In silico analysis was carried out using Ingenuity Pathways Analysis. In mammary gland tissue of LP rats at d21 of life a prominent upregulation of WT1 and CDKN1A (p21) expression was observed. Differentially regulated genes were associated with the extracellular regulated kinase (ERK)-1/-2 pathway. Western Blot analysis showed reduced levels of phosphorylated ERK-1/-2 in the mammary glands of the LP group at d21. To identify possible changes in circulating steroid levels, serum LC-Tandem mass-spectrometry was performed. LP rats showed higher serum progesterone levels and an increased corticosterone/dehydrocorticosterone-ratio at d28.Conclusions/SignificanceOur data obtained from gene array analysis support the hypothesis that IUGR influences pubertal development of the rat mammary gland. We identified prominent differential regulation of genes and pathways for factors regulating cell cycle and growth. Moreover, we detected new pathways which appear to be programmed by IUGR.

研究背景
宫内生长受限（Intrauterine growth restriction, IUGR）被认为可诱发胎儿程序化改变，进而引发多个器官出生后的发育异常。现有研究证实，IUGR是晚年罹患代谢与心血管疾病的风险因素，同时观察到IUGR个体日后乳腺癌发病率升高，这一现象可能与乳腺发育通路的改变相关。本研究旨在借助生物信息学通路分析方法，在IUGR大鼠模型的转录组层面，解析IUGR诱导的乳腺发育复杂通路改变。

研究方法与主要结果
我们通过母体低蛋白（LP）饮食构建IUGR Wistar大鼠模型，分别在青春期导管形态发生的起始阶段（出生后第21天，d21）与结束阶段（出生后第28天，d28）采集其乳腺组织。结果显示，LP组大鼠在d28时乳腺体积更小，但未观察到明显形态学改变。我们采用安捷伦SurePrint基因芯片在d21与d28时间点检测到乳腺内存在多个差异表达基因，并通过Ingenuity通路分析（Ingenuity Pathways Analysis）开展计算机模拟分析。
在出生后第21天的LP组大鼠乳腺组织中，我们观察到WT1与CDKN1A（p21）的表达显著上调。差异表达基因富集于细胞外调节蛋白激酶（ERK）-1/-2通路。蛋白质免疫印迹（Western Blot）分析显示，LP组大鼠d21时的乳腺组织中磷酸化ERK-1/-2水平降低。为检测循环类固醇激素水平的变化，我们采用血清液相色谱-串联质谱法进行检测，结果发现LP组大鼠在d28时血清孕酮水平升高，且皮质酮/脱氢皮质酮比值增加。

研究结论与意义
本研究通过基因芯片分析获得的数据支持了"IUGR可影响大鼠乳腺的青春期发育"这一假说。我们鉴定出调控细胞周期与生长的基因及通路存在显著差异表达，同时还发现了一批由IUGR程序化调控的全新通路。