Effect of Hypoxia in Severe Preeclampsia through Epigenetic Regulation. Homo sapiens

Though the pathophysiology of preeclampsia (PE) is unclear worldwide, placental hypoxia has been implicated in the pathologic processes of PE.In this study, we profiled the transcriptome in BeWo and JEG-3 cells cultured in hypoxic condition or normal ones based on the RNA sequencing dataset. After filtered the low-quality ones, the RNA readers was aligned to human genome hg19 by TopHat and then assembled by Cufflinks. The expression value of each transcript was calculated and consequently differentially expressed genes were screened out. CD39 and ZDHHC14 were found to have both different mRNA in hypoxic cells and abnormal methylation level in severely preeclamptic placentas. The mRNA expression of CD39 and ZDHHC14 in placental tissues were analyzed using qRT-PCR. The differential methylated regions (DMRs) of CD39 and ZDHHC14 were confirmed by MassARRY EppiTYPER. The effect of hypoxia on trophoblast cells was detected with western blotting and enzyme linked immunosorbent assay. The results showed CD39 and ZDHHC14were significantly lower in severe PE placenta, hypoxia significantly reduced the expression of CD39 and ZDHHC14 and the secretion of CD39 in trophoblast cells. Therefore, we believed hypoxia plays an important role in the processes of severe PE via decreasing the expression of CD39 and ZDHHC14 by changing their methylation level in placenta. Overall design: mRNA profiles of three cell lines at five stages and two different conditions were generated by deep sequencing, using HiSeq 2500.

尽管目前全球范围内对先兆子痫（preeclampsia, PE）的病理生理机制尚未完全阐明，但胎盘缺氧已被证实参与PE的病理进程。本研究基于RNA测序（RNA sequencing）数据集，对缺氧培养与正常培养条件下的BeWo细胞及JEG-3细胞开展转录组谱分析。过滤低质量数据后，使用TopHat工具将RNA测序读段比对至人类基因组hg19，随后通过Cufflinks完成转录本组装。计算各转录本的表达量后，进一步筛选出差异表达基因（differentially expressed genes）。研究发现，CD39与ZDHHC14在缺氧细胞中存在mRNA表达差异，且在重度先兆子痫患者的胎盘组织中呈现异常甲基化水平。本研究采用qRT-PCR分析了胎盘组织中CD39与ZDHHC14的mRNA表达水平，通过MassARRAY EpiTYPER验证了CD39与ZDHHC14的差异甲基化区域（differentially methylated regions, DMRs）。采用蛋白质印迹法（western blotting）与酶联免疫吸附实验（enzyme linked immunosorbent assay, ELISA）检测了缺氧对滋养层细胞的影响。结果显示，重度PE胎盘组织中CD39与ZDHHC14的表达量显著降低；缺氧可显著下调滋养层细胞中CD39与ZDHHC14的表达，并减少CD39的分泌。因此，我们认为缺氧可通过改变胎盘组织中CD39与ZDHHC14的甲基化水平，下调二者的表达，从而在重度PE的病理进程中发挥重要作用。总体实验设计：采用HiSeq 2500测序平台进行深度测序，获取3种细胞系在5个阶段及2种不同培养条件下的mRNA转录组谱。