差异表达微RNA作为多囊卵巢综合征生物标志物的研究

doi:10.3969/j.issn.1674-8115.2021.11.005

摘要/Abstract

摘要：

目的·探索可作为多囊卵巢综合征（polycystic ovary syndrome，PCOS）生物标志物的微RNA（microRNA，miRNA）及其在PCOS发病机制中的生物学意义。方法·选取上海交通大学医学院附属仁济医院2019年1月—10月收治的5例PCOS患者为观察组，另选取5例体检健康女性作为对照组。采集2组的颗粒细胞，用TRIzol法从中提取RNA，利用TruSeq Small RNA Library Prep试剂盒构建miRNA-seq文库，文库质检合格后用NextSeq500进行高通量测序，对测序结果进行生物信息学数据分析。采用RT-qPCR实验检验候选miRNA标志物是否存在差异表达。结果·分析获得颗粒细胞中20条差异表达miRNA（均P<0.05），其中miR-196a-5p、miR-10a-5p和miR-451a表达显著上调，miR-877-5p和miR-2355-5p表达显著下调，这些差异表达miRNA及其靶基因可能参与PCOS发生发展的转录调控机制。结论·差异表达miRNA具有作为PCOS生物标志物的潜能，并参与PCOS的发生发展。

关键词: 微RNA, 生物标志物, 多囊卵巢综合征, 基因表达, 靶基因

Abstract:

Objective·To explore microRNAs (miRNAs) as biomarkers of polycystic ovary syndrome (PCOS) and the biological significance in the pathogenesis of PCOS.

Methods·Five patients with PCOS from January 2019 to October 2019 in Renji Hospital, Shanghai Jiao Tong University School of Medicine were selected as the observation group, and 5 healthy women were selected as the control group. Granulosa cells from 2 groups were collected and RNA was extracted by TRIzol method. TruSeq Small RNA Library Prep Kit was used to construct miRNA-Seq library. After the quality inspection of the library, NextSeq500 was used for high-throughput sequencing. Bioinformatics data analysis was performed on the sequencing results. RT-qPCR was used to verify the differential expression of candidate miRNA markers.

Results·We finally got 20 differentially expressed miRNAs (all P<0.05). Among them, miR-196a-5p, miR-10a-5p and miR-451a were significantly up-regulated, while miR-877-5p and miR-2355-5p were significantly down-regulated. These differentially expressed miRNAs and their target genes may be involved in the transcriptional regulation mechanism of PCOS occurrence and development.

Conclusion·Differentially expressed miRNAs have potential as PCOS biomarkers and are involved in the occurrence and development of PCOS.

Key words: microRNA (miRNA), biomarker, polycystic ovary syndrome, gene expression, target gene

中图分类号:

R711.75

王昱欢, 丁奕岑, 蔡瑶雨, 康亚妮. 差异表达微RNA作为多囊卵巢综合征生物标志物的研究[J]. 上海交通大学学报(医学版), 2021, 41(11): 1429-1435.

Yu-huan WANG, Yi-cen DING, Yao-yu CAI, Ya-ni KANG. Study on differentially expressed microRNA as a biomarker of polycystic ovary syndrome[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(11): 1429-1435.

图/表 5

表1 miRNA引物序列（5′→3′）

Tab 1 miRNA primer sequence（5′→3′）

Primer		Sequence 5′→3′
miRNA	hsa-miR-196a-5p	GGGTAGGTAGTTTCATGTT
	hsa-miR-10a-5p	GGGTACAGTATAGATGA
	hsa-miR-451a	GGGAAACCGTTACCATTAC
	hsa-miR-877-5p	GGGGTAGAGGAGATGGC
	hsa-miR-2355-5p	GGGATCCCCAGATACAAT
	Adaptor	GATCGTCGGACTGTAGAACT

图1 miRNA差异分析的结果Note： A. Intersection of edgeR, DESeq2 and limma analysis. B. Volcano map of differential analysis. C. Hierarchical clustering heatmap of miRNA with differential expression.

Fig 1 Results of miRNA differential analysis

图2 靶基因的功能富集分析结果Note： A. GO enrichment analysis. B. KEGG enrichment analysis.

Fig 2 Results of functional enrichment analysis of target genes

图3 利用RT-qPCR实验验证5个miRNA候选生物标志物的基因表达结果Note： ①P=0.005, ②P=0.009, ③P=0.041, ④P=0.002, ⑤P=0.008.

Fig 3 RT-qPCR validation of 5 miRNA candidate markers

图4 PCOS疾病发生发展的转录调控机制

Fig 4 Transcriptional regulatory mechanism of PCOS occurrence and development

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