早发型重度子痫前期血浆外泌体miRNAs表达谱分析

Analysis of plasma exosomal miRNAs expression profile in early-onset severe preeclampsia

目的通过比较早发型重度子痫前期(EOPE)患者与正常妊娠妇女血浆外泌体源性miRNAs表达谱的差异,初步探讨EOPE的发病机制。方法收集于我院产检并最终诊断为EOPE的孕妇血浆(EOPE组,n=4)及正常妊娠妇女血浆(对照组,n=4);对外泌体进行鉴定,提取血浆外泌体中的miRNAs进行测序,对差异表达的miRNAs进行生物信息学分析。分别利用3个靶基因预测网站进行靶基因预测,利用Venn图绘制数据库所预测的靶基因交集。使用在线分析网站DAVID分析2组差异表达蛋白中的GO功能注释结果,然后进行KEGG通路富集分析。用RT-PCR检测胎盘及血浆外泌体miRNAs水平。结果检测到的颗粒直径为30~180 nm,峰值为112.8 nm;外形为茶状或椭圆形;2组均显著表达TSG101和CD63。测序结果显示:与对照组相比,EOPE组中共筛查出27个有显著表达差异的miRNAs,其中15个表达上调,12个表达下调;表达差异最显著的上调miRNA为miR-152。在线数据库网站预测所有差异表达miRNAs靶基因共8414个,经生物信息学分析显示其靶基因在补体、凝血级联、MMPs信号通路及TLR4信号通路等10个信号通路中富集程度最高。RT-PCR检测发现,与对照组相比,EOPE组中miR-152在胎盘及血浆外泌体中均表达上调(P<0.05)。结论EOPE患者血浆外泌体中miRNAs表达谱发生显著改变,这些差异表达的miRNAs可能参与了子痫前期的发生与发展,同时miR-152作为子痫前期的关键miRNA值得进一步研究。

Objective To investgate the pathogenesis of early-onset severe preeclampsia(EOPE)by comparing the expression of plasma exosomes-derived miRNAs from patients with EOPE and normal pregnant women.Methods The plasma samples from pregnant women who underwent antenatal examination and finally diagnosed as EOPE(the EOPE group,n=4)and normal pregnant women(the control group,n=4)in our hospital were collected.The identification of exosomes were conducted and the miRNAs from plasma exosomes were extracted for sequencing,and bioinformatics analysis was performed on differentially expressed miRNAs.Three target gene prediction websites were used for the prediction of target genes,and the target gene intersection predicted by the database was drawn by the Venn map.The annotation results of GO function in the two groups of differentially expressed proteins were analyzed by the online analysis website DAVID and the KEGG pathway enrichment analysis was carried out.The levels of miRNAs in placenta and plasma exosomes were detected by RT-PCR.Results The detected particle was 30 to 180 nm in diameter,with the peak value of 112.8 nm,and the shape was tea-like or oval.TSG101 and CD63 were significantly expressed in both two groups.The sequencing results showed that a total of 27 differentially expressed miRNAs were screened in the EOPE group compared with the control group,of which 15 were up-regulated and 12 were down-regulated,and miR-152 was the most significantly up-regulated miRNA.A total of 8414 differentially expressed miRNAs target genes were predicted by online database websites,and bioinformatics analysis showed that their target genes were most enriched in 10 signaling pathways,including complement and coagulation cascade,MMPs signaling pathway and TLR4 signaling pathway.The RT-PCR showed that the expression of miR-152 in placenta and plasma exosomes of the EOPE group was up-regulated compared with the control group(P<0.05).Conclusion The expression profile of miRNAs in plasma exosomes of EOPE patients changed significantly,and these differentially expressed miRNAs may be involved in the occurrence and development of preeclampsia.miR-152 is a key miRNA of preeclampsia,which deserves further study.