Ifitm1基因在小鼠卵泡发育与排卵过程中的功能及相关调控机制
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作者:
作者单位:

华中农业大学动物科学技术学院,武汉430070

作者简介:

熊昊,E-mail:1300928242@qq.com

通讯作者:

李凤娥,E-mail:lifener@mail.hzau.edu.cn

中图分类号:

S852.2

基金项目:

武汉市生物育种关键技术攻关及新品种培育科技重大专项(2022021302024853);湖北省科技重大专项(2021ABA005)


Function and related regulatory mechanisms of Ifitm1 gene in murine follicular development and ovulation
Author:
Affiliation:

College of Animal Science and Technology,Huazhong Agricultural University,Wuhan 430070,China

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    摘要:

    为深入了解哺乳动物卵泡发育的机制,对小鼠卵巢颗粒细胞中干扰素诱导的跨膜蛋白1(interferon-induced transmembrane protein 1,Ifitm1)基因进行超表达和抑制表达分析,通过流式细胞术、荧光定量PCR、EdU法及western blot分析Ifitm1对小鼠颗粒细胞生长的影响及对小鼠排卵相关基因表达的调控作用,并用相关通路抑制剂处理颗粒细胞,探究Ifitm1影响小鼠卵泡发育及排卵的相关机制。结果显示,在小鼠卵巢颗粒细胞中成功地超表达和抑制表达了Ifitm1基因,干扰 Ifitm1基因表达使细胞周期蛋白Ccnd1表达降低了63.5%,G0/G1期细胞占比也下降,使细胞阻滞在G2/M期,从而抑制颗粒细胞增殖;干扰Ifitm1基因还导致排卵标记基因LhrEregCyp19a1表达水平提高了1.95~6.76倍(P<0.05),并抑制PI3K/AKT信号通路上关键蛋白p-AKT(Ser473)的表达,而阻断PI3K/AKT信号通路后再抑制Ifitm1基因表达,LhrEregCyp19a1的mRNA水平则未出现明显改变,说明Ifitm1基因通过抑制PI3K/AKT信号通路活性影响排卵。以上结果表明,Ifitm1基因通过PI3K/AKT通路介导在小鼠颗粒细胞生长以及卵泡排卵过程中发挥重要作用。

    Abstract:

    In order to investigate the regulatory mechanisms of the interferon-induced transmembrane protein 1 gene (Ifitm1) in mammalian follicle development, Ifitm1 was overexpressed and suppressed in mouse granulosa cells (mGCs). The effects of Ifitm1 on the growth of mGCs were detected by flow cytometry, qRT-PCR and EdU method, the effects of Ifitm1 on the expression of ovulation-related genes in mice were detected by qRT-PCR and Western blot, and the mGCs were treated with related pathway inhibitors to investigate the mechanisms by which Ifitm1 affectes follicle development and ovulation in mice. The results showed that the Ifitm1 gene was successfully overexpressed and suppressed in mGCs. Further studies revealed that interference with Ifitm1 gene expression reduced the expression of cell cycle protein Ccnd1 by 63.5%, decreased the percentage of cells in G0/G1 phase, blocked the cells in G2/M phase, thus inhibiting the proliferation of granulosa cell. Ifitm1 interference also resulted in 1.95-6.76 fold increase in the expression levels of the ovulation marker genes LhrEreg and Cyp19a1P<0.05), and inhibited the expression of p-AKT (Ser473), a key protein in the PI3K/AKT signalling pathway, blocked the PI3K/AKT signalling pathway, and then inhibited the expression of Ifitm1 gene, but the mRNA levels of LhrEreg and Cyp19a1 remained unchanged in mGCs, suggesting that the Ifitm1 gene affects ovulation through inhibiting the activity of PI3K/AKT signalling pathway. In summary, Ifitm1 plays an important role in mGC growth and follicular ovulation through the PI3K/AKT pathway.

    表 2 定量扩增目的基因的引物Table 2 qRT-PCR primers for amplifying targeted gene
    表 1 寡核苷酸序列Table 1 Oligo sequences
    图1 小鼠颗粒细胞Ifitm1基因超表达和抑制表达Fig.1 Overexpression and suppression of Ifitm1 gene in mGCs
    图2 Ifitm1基因超表达或抑制表达后小鼠卵巢颗粒细胞周期Fig.2 Cell cycle of mGCs with overexpression or suppression of Ifitm1 gene
    图3 Ifitm1基因抑制表达后小鼠卵巢颗粒细胞的增殖和活力Fig.3 Cell proliferation and viability in mGCs treated with si-Ifitm1
    图4 超表达或抑制表达Ifitm1基因后卵巢颗粒细胞中排卵相关基因表达Fig.4 Ovulation-related gene expression in mGCs treated with pCMV-HA-Ifitm1 or si-Ifitm1
    图5 Ifitm1基因通过PI3K/AKT通路影响卵巢颗粒细胞中排卵相关基因表达Fig.5 Ifitm1 gene affects ovulation-related gene expression via PI3K/AKT pathway in mGCs
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熊昊,赵月秀,陈大可,陈亚茹,陈高贵,李凤娥.Ifitm1基因在小鼠卵泡发育与排卵过程中的功能及相关调控机制[J].华中农业大学学报,2023,42(4):199-206

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  • 收稿日期:2022-05-19
  • 在线发布日期: 2023-08-30
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