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

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

  • Reprod Biomed Online. 2022 May;44(5):803-816. doi: 10.1016/j.rbmo.2022.01.016.
Xiao Xu 1 Hao-Ran Shen 1 Min Yu 1 Mei-Rong Du 2 Xue-Lian Li 3
Affiliations

Affiliations

  • 1 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.
  • 2 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. Electronic address: mrdu@fudan.edu.cn.
  • 3 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. Electronic address: xllifc@fudan.edu.cn.
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.

Keywords

Granulosa cell; IMP2; Let-7i; Oestradiol biosynthesis; Polycystic ovary syndrome; Proliferation.

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