11p15.5区域甲基化状态对胎盘印迹基因表达、滋养细胞增殖的调控及其对单合子双胎宫内发育不一致的影响

The abnormal expressions of imprinting genes which located at 11p15.5 region are related to the aberration of trophoblast proliferation and probably lead to the disorder of fetal development. DNA methylation plays an important role in regulating the gene expression, but its relation with abnormal expressions of imprinting genes and aberration of trophoblast proliferation are still unclear. Since the polymorphism of DNA background of individual sample in singleton study, the former results based on singleton study may not fully reflect the effect of methylation modification on trophoblast proliferation and fetal development. Our preliminary experiments indicated that the status of proliferation of trophoblast were different in discordant monozygotic twins who had similar DNA sequence and intrauterine environment,and this alteration was probably related to the functions of imprinting genes located in 11p15.5 region. Thereofore, we hypothesized that the methylation modifications in the 11p15.5 region might regulate the imprinting genes and then influence the proliferation of trophoblast, which may be one of the etiologies of discordant development in monozygotic twins. In the subsequent proposal, we plan to use the techniques of DNA methylation pyrosequencing, real-time PCR, flow cytometry to compare: (1)The DNA methylation status and the imprinting genes' expressions of 11p15.5 region; (2) The proliferation and apoptosis status of trophoblasts in the placenta of discordant twins. Based on these results, we will further explore the impacts of methylation modification of important loci and imprinting genes on the proliferation status of trophoblasts and possible mechanisms in in vitro cell culture by the methods of methylation modification and cell transfection. Our reasearch may provide important clues of prevention and treatment for fetal growth disorder.

11p15.5区域印迹基因异常表达可致胎盘滋养细胞增殖失常并引起胎儿发育异常。DNA甲基化是调控基因表达的重要方式，但与印迹基因异常表达及滋养细胞增殖失调的关系尚不清楚。以往采用单胎研究，因个体间DNA序列的多态性，结果难以准确反映甲基化修饰的改变对滋养细胞增殖及胎儿发育的影响。我们采用DNA序列及宫内环境相同的单合子双胎进行前期研究发现：11p15.5区域印迹基因的表达与发育不一致双胎胎盘滋养细胞增殖有关。由此提出：11p15.5区域甲基化修饰的改变可能引起印迹基因表达异常，继而影响滋养细胞的增殖，最终导致两胎发育不一致。本研究拟采用焦磷酸测序、定量PCR、流式细胞等技术，比较发育不一致单合子双胎胎盘11p15.5区域甲基化、印迹基因表达及滋养细胞增殖间的差别，通过甲基化修饰、细胞转染等手段阐明该区域甲基化状态对印迹基因表达及滋养细胞增殖的作用机制，为防治胎儿发育异常提供重要依据。

以往研究认为胎盘份额不均及脐带插入异常是MCDA双胎sIUGR的主要病因，近年有文献报道，表观遗传学的改变在单合子双胎的发育差异上有重要作用。我们前期研究发现11p15.5区域印迹基因的表达与发育不一致双胎胎盘滋养细胞增殖有关。本项目采用单合子双胎sIUGR作为研究对象，探讨胎盘11p15.5区域印迹基因PHLDA2启动子甲基化修饰及其基因表达、HERVWEl基因调控序列甲基化状态、基因组启动子甲基化、LINE-1 DNA甲基化等与单合子双胎sIUGR的关系。结果显示，基因组启动子部分位点甲基化水平减低可能与单合子双胎sIUGR发生有关；去甲基化修饰上调PHLDA2的表达，影响滋养细胞增殖、凋亡及细胞周期调控，甲基化修饰异常可能影响胎盘胎儿生长发育；且HERVWEl表达与5’端调控区域的甲基化有关，提示体重轻的胎儿胎盘滋养细胞融合和增殖功能可能出现代偿性增强的趋势。研究表明11p15.5区域甲基化修饰的改变可能引起印迹基因表达异常，继而影响滋养细胞的增殖，最终导致双胎发育不一致。