微小 RNA let-7i 通过直接靶向 IMP2 抑制颗粒细胞黄体细胞增殖和雌二醇生物合成。

MicroRNA let-7i inhibits granulosa-luteal cell proliferation and oestradiol biosynthesis by directly targeting IMP2.

机构信息

Obstetrics and Gynecology Hospital, Fudan University Shanghai, People's Republic of China; Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Obstetrics and Gynecology Hospital, Fudan University Shanghai, People's Republic of China.

出版信息

Reprod Biomed Online. 2022 May;44(5):803-816. doi: 10.1016/j.rbmo.2022.01.016. Epub 2022 Feb 3.

DOI:10.1016/j.rbmo.2022.01.016

PMID:35339367

Abstract

RESEARCH QUESTION

Increased granulosa cell division is associated with abnormal folliculogenesis in polycystic ovary syndrome (PCOS). Lethal-7i microRNA (let-7i) may play an important role in the follicular development and granulosa cell growth; therefore is let-7i involved in PCOS pathogenesis?

DESIGN

The expression of let-7i was measured in granulosa-luteal cells (GLC) from women with or without PCOS. A human granulosa cell line, KGN, was used for the functional study. Mimics and inhibitors of let-7i, lentiviruses expressing insulin-like growth factor 2 mRNA binding protein (IMP2), and small-interfering RNAs were transfected into KGN cells. KGN cell proliferation was determined by 5-ethynyl-2'-deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assays. The cell cycle and apoptosis were assessed by propidium iodide-annexin V (PI-A) staining and fluorescence-activated cell sorting. Oestradiol concentration was determined by enzyme-linked immunoassay. Bioinformatics analysis and luciferase reporter assay were applied to confirm the let-7i target genes.

RESULTS

The study showed that let-7i was down-regulated in PCOS GLC (P = 0.001). Mimics of let-7i inhibited KGN proliferation (P = 0.001), and decreased aromatase expression (P = 0.030) and oestradiol production (P = 0.029), whereas let-7i inhibitors had the opposite effect. Bioinformatics analysis and quantitative real-time (qRT) PCR identified IMP2 as a target of let-7i (P = 0.021). qRT-PCR and western blot analysis indicated that IMP2 was up-regulated in GLC in women with PCOS (P = 0.001 and P = 0.044), and IMP2 expression was suppressed by let-7i in KGN cells (P < 0.001). Luciferase reporter assay results (P = 0.002), combined with the rescue assay, confirmed that let-7i inhibited KGN cell proliferation and reduced oestradiol concentration by directly targeting IMP2.

CONCLUSIONS

let-7i was down-regulated in PCOS GLC. Overexpression of let-7i inhibited KGN cell proliferation and decreased oestradiol production in an IMP2-dependent manner, providing a new molecular mechanism for PCOS.

摘要

研究问题

在多囊卵巢综合征（PCOS）中，颗粒细胞分裂增加与异常卵泡发生有关。致死-7i 微 RNA（let-7i）可能在卵泡发育和颗粒细胞生长中发挥重要作用；因此，let-7i 是否参与了 PCOS 的发病机制？

设计

测量有无 PCOS 的女性颗粒细胞-黄体细胞（GLC）中的 let-7i 表达。用人颗粒细胞系 KGN 进行功能研究。转染 let-7i 的模拟物和抑制剂、表达胰岛素样生长因子 2 mRNA 结合蛋白（IMP2）的慢病毒和小干扰 RNA 进入 KGN 细胞。通过 5-乙炔基-2'-脱氧尿苷（EdU）和细胞计数试剂盒-8（CCK-8）测定 KGN 细胞增殖。通过碘化丙啶-膜联蛋白 V（PI-A）染色和荧光激活细胞分选评估细胞周期和凋亡。通过酶联免疫吸附试验测定雌二醇浓度。应用生物信息学分析和荧光素酶报告基因检测来确认 let-7i 的靶基因。

结果

研究表明，PCOS GLC 中 let-7i 下调（P=0.001）。let-7i 的模拟物抑制 KGN 增殖（P=0.001），并降低芳香化酶表达（P=0.030）和雌二醇产生（P=0.029），而 let-7i 抑制剂则有相反的效果。生物信息学分析和实时定量（qRT）PCR 鉴定 IMP2 为 let-7i 的靶基因（P=0.021）。qRT-PCR 和 Western blot 分析表明，PCOS 患者的 GLC 中 IMP2 上调（P=0.001 和 P=0.044），let-7i 在 KGN 细胞中抑制 IMP2 表达（P<0.001）。荧光素酶报告基因检测结果（P=0.002），结合挽救试验，证实 let-7i 通过直接靶向 IMP2 抑制 KGN 细胞增殖并降低雌二醇浓度。