孤立性侧脑室扩张胎儿孕妇羊水外泌体miRNA差异表达谱

doi:10.12122/j.issn.1673-4254.2024.11.24

摘要/Abstract

摘要：

目的探讨孤立性侧脑室扩张胎儿孕妇羊水外泌体中的微小RNA（miRNA）的差异表达，对其靶基因进行预测分析。方法收集2021年9月~2024年5月在南方医科大学南方医院产前超声诊断为胎儿中度孤立性侧脑室扩张的孕妇羊水样本9例，和由于高龄或唐筛高风险行羊水穿刺的正常胎儿对照组的孕妇羊水样本8例。分离羊水外泌体，运用miRNA测序技术筛选两组羊水外泌体中的差异表达miRNA，并从中挑选3个在对照和样本组中的表达差异显著，且文献报道参与的信号通路和侧脑室关联的miRNA进行qPCR验证测序结果。对显著差异表达的TOP 40个miRNA进行靶基因预测及功能分析（GO）和信号通路分析（KEGG）。通过双荧光素酶报告基因技术验证miR-122-5p对预测靶基因AKT3和CCDC88C的调控。结果与对照组相比，侧脑室扩张组中存在272个显著差异表达miRNA，其中表达上调的有43个，表达下调的有229个。GO分析发现靶基因主要功能与转录因子结合、转运蛋白活性、神经系统过程、跨膜运输等有关。KEGG通路分析发现富集显著的功能通路包括有MAPK信号通路、Wnt信号通路、配体-受体神经活性互作和细胞因子-细胞因子受体互作等。qPCR验证结果发现，与对照组相比，miR-122-5p表达显著下降（P<0.05），与测序结果一致；而let-7b-5p的表达病例组较对照组下调，与测序结果相反。双荧光素酶报告基因检测结果显示miR-122-5p不调控AKT3和CCDC88C的表达。结论羊水外泌体中高丰度差异表达的miRNAs可能通过参与MAPK信号通路、PI3K-Akt信号通路、Wnt信号通路和cGMP-PKG通路在胎儿侧脑室扩张的发生发展中发挥作用。

关键词: 孤立性侧脑室扩张, 羊水, 外泌体, 微小RNA

Abstract:

Objective To investigate the role of miRNAs in maternal amniotic fluid exosomes in development of isolated ventriculomegaly (VM) in fetuses. Methods Amniotic fluid samples were collected from 9 cases of moderate isolated VM and 8 normal control cases to extract exosomal miRNA, and miRNA sequencing technique was used to identify differentially expressed miRNAs between the two groups. Three miRNAs with significant differential expression between the two groups, whose high expression was associated with VM, were selected for verification with RT-qPCR. Dual luciferase reporter assays were used to verify the regulatory effect of miR-122-5p on its predicted target genes AKT3 and CCDC88C. Gene ontology (GO) and KEGG pathway analyses were performed to explore the possible roles of the top 40 significant differential miRNAs in the pathophysiology of VM. Results We identified a total of 272 differentially expressed miRNAs in VM cases, including 43 up-regulated and 229 down-regulated miRNAs. The target genes of these differential miRNAs were associated with DNA and transcription factor binding, transmembrane transporter and nucleic acid binding transcription factor activity, and cell developmental process. These miRNAs were mostly enriched in the MAPK, cGMP-PKG and Wnt signaling pathways. Verification with RT-qPCR showed that miR-122-5p expression level was significantly lower in VM group than in the control group (P<0.05), which was consistent with miRNA sequencing results; let-7b-5p expression level was significantly lower in VM group, which was contrary to miRNA sequencing result. Dual luciferase reporter assays showed that miR-122-5p was not capable of regulating AKT3 or CCDC88C expressions. Conclusions The highly abundant differentially expressed miRNAs in maternal amniotic fluid exosomes play important roles in the occurrence of fetal VM possibly by regulating the MAPK, PI3K-Akt, Wnt and cGMP-PKG signaling pathways.

Key words: fetal isolated ventriculomegaly, amniotic fluid, exosomes, miRNA

李粉霞, 林浩生, 黎一琳, 朱雯倩, 孙元洁, 黄源, 裘毓雯, 秦霞, 常清贤. 孤立性侧脑室扩张胎儿孕妇羊水外泌体miRNA差异表达谱[J]. 南方医科大学学报, 2024, 44(11): 2256-2264.

Fenxia LI, Haosheng LIN, Yilin LI, Wenqian ZHU, Yuanjie SUN, Yuan HUANG, Yuwen QIU, Xia QIN, Qingxian CHANG. Differential expression profile of miRNAs in maternal amniotic fluid exosomes in fetuses with isolated ventriculomegaly[J]. Journal of Southern Medical University, 2024, 44(11): 2256-2264.

图/表 6

表1 miR-122-5p和ATK3， CCDC88C的合成序列

Tab.1 Synthesized sequences of miR-122, ATK3, and CCDC88C for dual luciferase reporter assays

Name	Sequence
miR-122-5p	3' gtT-TGTG-GTAACAGTGTGAGGt 5'
AKT3 3'UTR-WT	5' acATACACGCA-AAT-ACACTCCa 3'
AKT3 3'UTR-mut	5' actTtCtCcCt-AtT-tCtCaCgA 3'
CCDC88C 3'UTR-WT	5' agAAGATGAGTTGTCACACTCCc 3'
CCDC88C 3'UTR-mut	5' agtAGATGAGaTcTgAgAgTgCg 3'

图1 羊水外泌体鉴定

Fig.1 Identification of exosomes in maternal amniotic fluid. A: Exosome morphology under transmission electron microscope. B: Protein expressions of exosome markers CD9, CD63, CD81 and TSG101 detected by Western blotting. C: Particle size of the exosomes.

图2 羊水外泌体在侧脑室扩张样本和对照组中差异表达的miRNA的丰度热图

Fig.2 Heat map of the differentially expressed miRNAs between ventriculomegaly and control groups. Red and blue indicates up-regulated and down-regulated miRNAs, respectively.

图3 qPCR检测两组羊水外泌体中let-7b-5p,miR-122-5p和miR-146a-5p表达水平

Fig.3 Expression levels of let-7b-5p, miR-122-5p and miR-146a-5p in amniotic fluid exosomes from the two groups detected by RT-qPCR.

图4 miR-122-5p 的预测靶基因荧光素酶活性测定结果

Fig.4 Result of 3'UTR luciferase report system for assessing interactions between miRNA-122-5p and its predicted target genes. A: Luciferase assays with AKT3 3'UTR-WT/mut. B: Luciferase assays with CCDC88C 3'UTR-WT/mut.

图5 KEGG信号通路分析TOP40差异表达miRNA的靶基因功能

Fig.5 KEGG pathway analysis of the top 40 significant differential miRNAs in the exosomes for biological functions of their target genes.

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