南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (6): 801-810.doi: 10.12122/j.issn.1673-4254.2021.06.01

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miR-483-5p通过靶向FKBP4加重顺铂诱导的小鼠卵巢损伤

赵茴茴，古文清，潘文斌，张汉彬，帅 领，刁瑞英，汪丽萍   

  • 出版日期:2021-06-20 发布日期:2021-07-05

miR-483-5p aggravates cisplatin-induced premature ovarian insufficiency in rats by targeting FKBP4

  • Online:2021-06-20 Published:2021-07-05

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摘要： 目的 探讨FKBP4蛋白在顺铂诱导的早发性卵巢功能不全（POI）中的作用及其机制研究。方法 采用ITRAQ技术测定4对顺铂诱导的小鼠POI模型组及生理盐水对照组卵巢组织的差异蛋白。使用TargetScan软件进行靶点预测，利用qRT-PCR和Western blot方法确定顺铂诱导组及生理盐水对照组miR-483-5p及FKBP4的表达水平。收集POI患者及正常患者血清各 6 例，并利用 qRT-PCR 检测 miR-483-5p 表达水平。利用细胞转染及双荧光素酶实验验证 miR-483-5p 和 FKBP4 的关系。利用转染和/或顺铂分别处理原代颗粒细胞及KGN细胞系（人颗粒细胞系），将细胞分为对照（NC）组、miR-483-5p组、miR-483-5p+顺铂处理组和miR-483-5p+FKBP4+顺铂处理组。以此验证miR-483-5p、FKBP4在顺铂处理的颗粒细胞中的作用；利用 Cre-loxp 系统构建卵母细胞特异性过表达 miR-483-5p 及同窝未过表达的转基因小鼠，用免疫荧光、原位杂交及ELISA检测卵巢功能。结果 与生理盐水组相比，顺铂诱导的POI小鼠的卵巢中FKBP4表达下降（P<0.05）。靶点预测软件发现miR-483-5p的潜在靶点是FKBP4，并且较生理盐水对照组，它在顺铂诱导的POI小鼠的卵巢、血清以及POI患者的血清中均高水平表达（P<0.01）。体外实验进一步证实FKBP4是miR-483-5p的作用靶点。人颗粒细胞系及小鼠原代颗粒细胞过表达FKBP4缓解了顺铂联合miR-483-5p过表达引起的细胞凋亡（n=5，P<0.05）。与同窝未过表达的小鼠比较，卵母细胞过表达miR-483-5p的小鼠顺铂诱导造成的卵巢损伤更严重。结论 miR-483-5p/FKBP4在顺铂诱导的POI中发挥作用。顺铂诱导的卵巢损伤过程可能与上调的miR-483-5p靶向作用FKBP4，导致的FKBP4表达下降有关。miR-483-5p上调可能增加卵巢对顺铂药物的敏感性，使卵巢功能降低。检测血清miR-483-5p可以用来预测POI的发生和发展。

关键词: 卵巢功能不全；顺铂；卵巢损伤；miR-483-5p；FKBP4

Abstract: Objective To investigate the role of FKBP4 protein in cisplatin-induced premature ovarian insufficiency (POI). Methods We performed ITRAQ assay of the ovarian tissues from 4 mice with cisplatin-induced POI and 4 control mice, and identified FKBP4 as a significantly down-regulated protein in the oocytes and granulosa cells following cisplatin treatment. TargetScan software was used for target analysis of FKBP4, and qRT-PCR and Western blotting were used to verify the expression levels of miR-483-5p and FKBP4 in the mouse models. Serum samples were collected from patients with POI and healthy women for detecting miR-483-5p level with qRT-PCR. Cell transfection and dual-luciferase assay were performed to determine the relationship between miR-483-5p and FKBP4. In primary granulosa cells and KGN cells, we examined the effect of miR-483-5p alone, miR-483-5p and cisplatin, and miR-483-5p combined with both cisplatin and FKBP4 on cell apoptosis. We also assessed ovarian function in a transgenic mouse model with ovarian miR-483-5p overexpression in comparison wigh wild-type mice using immunofluorescence assay, in situ hybridization and ELISA. Results Ovarian FKBP4 expression was significantly decreased in mice with cisplatin-induced POI. Analysis using TargetScan software indicated that FKBP4 was the potential target of miR-483-5p, which was highly expressed in the ovaries and serum of POI mice and in the serum of patients with POI. In vitro experiments further confirmed that FKBP4 was the target of miR-483-5p. In KGN and primary granulosa cells, FKBP4 overexpression significantly reduced cell apoptosis induced by both cisplatin and miR-483-5p overexpression (P= 0.0045 and 0.0177, respectively). In the transgenic mice with miR-483-5p overexpression in the oocytes, cisplatin induced more severe ovarian damages as compared with those in the wild-type mice. Conclusion miR-483-5p/FKBP4 is a new and important pathway in cisplatin-induced POI, in which cisplatin increases ovarian miR- 483-5p expression to result in targeted down-regulation of FKBP4. Up-regulation of miR-483-5p may increase ovarian sensitivity to cisplatin and cause severe ovarian dysfunction. Detection of serum miR-483-5p level may help to predict the occurrence and development of POI.

Key words: premature ovarian insufficiency; cisplatin; ovarian injury; miR-483-5p; FKBP4