PCOS患者卵泡液外泌体miRNA谱差异表达及生物信息学分析

Differential expression of exosomal microRNA in the follicular fluid of women with polycystic ovary syndrome and its bioinformatics analysis

目的检测多囊卵巢综合征(PCOS)患者卵泡液外泌体微小RNA(miRNA)的表达差异,探究其成为胚胎质量相关的潜在生物标志物的可能性。方法收集2017年9月至2018年2月在同济大学附属同济医院生殖医学中心接受卵胞浆内单精子注射(ICSI)助孕的女性不孕症患者[包括12例PCOS患者(PCOS组)和12例卵巢功能正常的不孕症患者(对照组)]的卵泡液。依据D3胚胎质量将两组卵泡分为优胚组(PCOS-1,n=11;对照-1,n=15)和非优胚组(PCOS-2,n=11;对照-2,n=15),将对应卵泡液等量混合形成4个25 ml卵泡液池。利用试剂盒提取卵泡液外泌体,使用Illumina HiSeq 2500平台检测外泌体miRNA表达量,构建miRNA差异表达谱。利用生物信息学方法(miRanda、miRDB和miRTarBase软件和KOBAS数据库)预测差异miRNA的靶基因,再对靶基因进行GO功能注释和KEGG通路分析;利用Cytoscape_v3.8.2软件进行互作网络分析并计算网络及各个节点的拓扑特性。结果(1)与优胚组相比,非优胚组有9个显著差异表达的外泌体miRNA,其中7个表达上调,2个表达下调(P<0.05且|-log2(Fold Change)|≥1)。预测靶基因后采用GO和KEGG、Cytoscape_v3.8.2分析发现,hsa-miR-200a-3p、hsa-miR-141-3p共同调控靶基因,主要富集在P13K-Akt、调节干细胞多能性信号通路,涉及蛋白结合、核酸结合等分子功能。(2)与PCOS-1组相比,PCOS-2组有120个显著差异表达的外泌体miRNA,其中57个表达上调,63个表达下调(P<0.01且|-log2(Fold Change)|≥2)。预测靶基因后采用GO和KEGG、Cytoscape_v3.8.2分析发现,hsa-miR-26b-5p、hsa-miR-483-5p、hsa-miR-218-5p、hsa-miR-320b、hsa-miR-320c等关键miRNA调控靶基因,主要富集在MAPK、P13K-Akt、FoxO、FAK、mTOR、Ras/MAPK胰岛素信号通路、AGE-RAGE信号通路以及癌症、细胞周期等信号通路,参与细胞代谢调控、细胞大分子代谢、细胞调控等生物过程,涉及离子结合、DNA结合、酶活性等分子功能。结论筛选所得的核心外泌体miRNA可能影响PCOS患者胚胎发育的过程,有可能成为预测胚胎质量的生物标志物。

Objective:To detect the expression of exosomal microRNA(miRNA)in the follicular fluid of women with polycystic ovary syndrome(PCOS)by using high throughput sequencing technique,and explore the possibility of it as the potential biomarkers related to embryo quality.Methods:The samples of follicular fluid were collected from infertile women,including 12 PCOS patients(PCOS group)and 12 infertile patients with normal ovarian function(control group),who received ICSI in the Reproductive Medicine Center of Tongji Hospital of Tongji University from September 2017 to February 2018.According to the Day 3 embryo quality,the samples of follicular fluid of the two groups were divided into the good-quality embryo group(PCOS-1 group,n=11;control-1 group,n=15)and non-good-quality embryo group(PCOS-2 group,n=11;control-2 group,n=15).The corresponding follicle fluids were mixed in equal amount to form four 25 ml follicle fluid pools.The exosomes were extracted by kit.The exosomal miRNA expressions were detected by using Ilumina HiSeq 2500 to construct miRNA differential expression profile.Bioinformatics technique(miRanda,miRDB and miRTarBase software and KOBAS datebase)was used to predict target genes of miRNA,and then GO function annotation and KEGG pathway analysis of target genes were carried out.Cytoscape_v3.8.2 software was used for interaction network analysis,and the topological characteristics of the network and each node were calculated.Results:(1)Compared with the good-quality embryo group,there were 9 differentially expressed exosomal miRNAs in the non-good-quality embryo group,of which 7 up-regulated and 2 down-regulated(P<0.05 and|-log2(fold change)|≥1).Go,KEGG and Cytoscape_v3.8.2 analysis found that hsa-miR-200a-3p and hsa-miR-141-3p co-regulated the target genes,which were mainly enriched in P13K-Akt and the signaling pathway regulating pluripotency stem of cells,involving protein binding,nucleic acid binding molecular functions.(2)Compared with PCOS-1 group,there were 120 differentially expressed exosomal miRNAs in PCOS-2 group,of which 57 were up-regulated and 63 were down-regulated(P<0.01 and|-log2(fold change)|≥2).Go,KEGG and Cytoscape_v3.8.2 analysis predicted that hsa-miR-26b-5p,hsa-miR-483-5p,hsa-miR-218-5p,hsa-miR-320b and hsa-miR-320c were the key miRNAs regulation target genes,which were mainly enriched in MAPK,P13K-Akt,FoxO,FAK,mTOR,Ras/MAPK insulin signaling pathway,AGE-RAGE signaling pathway,as well as cancer&cell cycle signaling pathways,and participated in cellular metabolic regulation,cell macromolecular metabolic,cell regulation and other cellular biological processes,involving ion binding,DNA binding,catalytic activity and other molecular functions.Conclusions:The screened core exosomal miRNAs may affect the embryo development of PCOS patients,and may become the biomarkers for predicting embryo quality.