采用下一代测序和实时定量聚合酶链反应鉴定子宫内膜异位症的候选 microRNA 标志物。

Identification of candidate microRNA markers of endometriosis with the use of next-generation sequencing and quantitative real-time polymerase chain reaction.

机构信息

Department of Medical Genetics, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.

出版信息

Fertil Steril. 2020 Jun;113(6):1232-1241. doi: 10.1016/j.fertnstert.2020.01.026.

DOI:10.1016/j.fertnstert.2020.01.026

PMID:32482255

Abstract

OBJECTIVE

To identify novel candidate diagnostic microRNA (miRNA) markers of endometriosis by means of an unbiased search with confirmation by means of targeted polymerase chain reaction (PCR).

DESIGN

Retrospective cohort.

SETTING

University teaching hospitals.

PATIENT(S): Women with endometriosis and control women, confirmed with the use of laparoscopy.

INTERVENTIONS(S): Diagnostic laparoscopy and blood sample.

MAIN OUTCOME MEASURE(S): Next-generation sequencing (NGS) and quantitative real-time PCR (qRT-PCR).

RESULT(S): Candidate miRNAs differentially expressed in women with endometriosis compared with control women were identified by means of NGS and selected for qRT-PCR. Plasma samples from another cohort of women with surgically confirmed endometriosis (n = 53) and disease-free control women (n = 53) were checked for hemolysis using spectrophotometry and the ratio of miR-23a and miR-451 by means of qRT-PCR. MicroRNA signatures were quantified by means of qRT-PCR in hemolysis-free plasma samples of case subjects (n = 25) and control subjects (n = 28) with the use of miRcury LNA miRNA. Circulating levels of eight miRNAs (miR-199a-3p, miR-143-3p, miR-340-5p, let-7b-5p, miR-21-5p, miR-17-5p, miR-20a-5p, and miR-103a-3p) were significantly lower in case subjects compared to control subjects. The sensitivity and specificity for individual miRNAs ranged from 0.36 to 1.00 and from 0.43 to 1.00, respectively, but when combined produced sensitivity and specificity of 0.92 and 0.86 with positive (PPV) and (NPV) predictive values of 0.85 and 0.92, respectively. However, combination of five miRNAs (miR-17-5p, miR-20a-5p, miR-199a-3p, miR-143-3p, and let-7b-5p) produced sensitivity and specificity of 0.96 and 0.79 with PPV and NPV of 0.80 and 0.96, respectively.

CONCLUSION(S): We conclude that a panel of candidate miRNAs was comparable to laparoscopy in distinguishing between women with endometriosis and control women.

摘要

目的

通过无偏搜索并通过靶向聚合酶链反应（PCR）进行确认，鉴定子宫内膜异位症的新型候选诊断微小 RNA（miRNA）标志物。

设计

回顾性队列。

地点

大学教学医院。

患者

子宫内膜异位症患者和经腹腔镜证实的对照妇女。

干预措施

诊断腹腔镜检查和血液样本。

主要观察指标

下一代测序（NGS）和实时定量 PCR（qRT-PCR）。

结果

通过 NGS 鉴定出与对照妇女相比在子宫内膜异位症妇女中表达差异的候选 miRNA，并通过 qRT-PCR 进行选择。使用分光光度法检查另一批经手术证实的子宫内膜异位症（n = 53）和无疾病对照妇女（n = 53）的血浆样本是否溶血，并通过 qRT-PCR 检查 miR-23a 和 miR-451 的比值。使用 miRcury LNA miRNA 在无溶血的病例组（n = 25）和对照组（n = 28）血浆样本中定量 miRNA 特征通过 qRT-PCR 进行。与对照组相比，病例组中 8 种 miRNA（miR-199a-3p、miR-143-3p、miR-340-5p、let-7b-5p、miR-21-5p、miR-17-5p、miR-20a-5p 和 miR-103a-3p）的循环水平显着降低。单独 miRNA 的灵敏度和特异性范围为 0.36 至 1.00，分别为 0.43 至 1.00，但当组合使用时，敏感性和特异性为 0.92 和 0.86，阳性（PPV）和（NPV）预测值分别为 0.85 和 0.92。然而，当组合使用 5 种 miRNA（miR-17-5p、miR-20a-5p、miR-199a-3p、miR-143-3p 和 let-7b-5p）时，敏感性和特异性为 0.96 和 0.79，PPV 和 NPV 分别为 0.80 和 0.96。