Identification of microRNAs that Regulate the MAPK Pathway in Human Cumulus Cells from PCOS Women with Insulin Resistance
Identification of microRNAs that Regulate the MAPK Pathway in Human Cumulus Cells from PCOS Women with Insulin Resistance
复制标题
鉴定调节患有胰岛素抵抗的 PCOS 女性人卵丘细胞 MAPK 通路的 microRNA
DOI：
10.1007/s43032-019-00086-5
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发表时间：
2020-03-01
影响因子：
2.9
通讯作者：
Xu, Bo
中科院分区：
文献类型：
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作者：
Hu, Mei-hong;Zheng, Sheng-xia;Xu, Bo
多囊卵巢综合征（PCOS）是最常见的妇科内分泌疾病之一，60%以上的PCOS患者存在不同程度的胰岛素抵抗（IR）。microRNAs（miRNAs）在人类卵丘细胞中转录后水平与PCOS IR相关的调节作用尚不清楚。在这项病例对照研究中，26例PCOS伴IR患者（PCOS-IR）和24例无IR患者（PCOS-control）入选。我们使用新一代测序技术确定差异表达的miRNA和mRNA，并通过定量实时聚合酶链反应（PCR）验证这些miRNA和mRNA。这些miRNA调节途径（例如，通过实时荧光定量PCR、western blot和荧光素酶活性检测，证实Rap 1b是miR-612的靶点。共鉴定出59个已知的miRNAs和617个差异表达基因，它们在PCOS-IR和PCOS对照卵丘细胞之间差异表达。此外，通过基因本体论（GO）和京都基因与基因组百科全书（KEGG）途径注释，分析了miRNAs及其靶基因在IR和PCOS病理生理中的潜在调控作用，并对MAPK活性等几个关键过程进行了富集。此外，基于负表达相关验证、双荧光素酶活性测定和miR-612模拟物转染后Rap 1b表达的减少，证明了MAPK通路的调节剂Rap 1b在PCOS-IR卵丘细胞中被miR-612直接抑制。提示卵巢卵丘细胞中miRNAs及其靶向通路可能在PCOS伴IR的病因学和病理生理学中发挥重要作用。
Polycystic ovary syndrome (PCOS) is one of the most common gynaecological endocrine disorders, and more than 60% of PCOS patients have varying degrees of insulin resistance (IR). The regulatory role of microRNAs (miRNAs) at post-transcriptional levels in human cumulus cells relating to IR in PCOS remains unclear. In this case-control study, 26 PCOS patients with IR (PCOS-IR) and 24 patients without IR (PCOS-control) were enrolled. We determined the differentially expressed miRNA and mRNA using next-generation sequencing technology, and these miRNAs and mRNAs were validated by quantitative real-time polymerase chain reaction (PCR). These miRNA regulating pathways (e.g., MAPK pathway) were analysed by bioinformatics analysis, and the Rap1b was demonstrated to be targeted by miR-612 based on quantitative real-time PCR, western blot and luciferase activity assay. A total of 59 known miRNAs and 617 differentially expressed genes were identified that differentially expressed between PCOS-IR and PCOS-control cumulus cells. Moreover, the potential regulating roles of miRNAs and their targeting genes in pathophysiology of IR and PCOS were analysed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation, and several key processes were enriched, such as MAPK activity. Furthermore, Rap1b, a regulator of the MAPK pathway, was demonstrated to be suppressed directly by miR-612 in PCOS-IR cumulus cells based on negative expression correlation validation, dual luciferase activity assay and reduction of Rap1b expression after miR-612 mimics transfection. Our results suggested that miRNAs and their targeted pathways in ovarian cumulus cells may play important roles in the aetiology and pathophysiology of PCOS with IR.