miR-99a-5p inhibits target gene FZD5 expression and steroid hormone secretion from goat ovarian granulosa cells

doi:10.1016/S2095-3119(21)63766-8

Journal of Integrative Agriculture ›› 2022, Vol. 21 ›› Issue (4): 1137-1145.DOI: 10.1016/S2095-3119(21)63766-8

收稿日期:2020-10-22 接受日期:2021-06-04 出版日期:2022-04-01 发布日期:2021-06-04

miR-99a-5p inhibits target gene FZD5 expression and steroid hormone secretion from goat ovarian granulosa cells

ZHU Lu^{1, 2*}, JING Jing^{1, 2*}, QIN Shuai-qi^{1, 2}, LU Jia-ni^{1, 2}, ZHU Cui-yun^{1, 2}, ZHENG Qi^{1, 2}, LIU Ya^{1, 2}, FANG Fu-gui^{1, 2}, LI Yun-sheng^{1, 2}, ZHANG Yun-hai1,², LING Ying-hui^{1, 2}

¹ College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, P.R.China
² Local Animal Genetic Resources Conservation and Biobreeding Laboratory of Anhui Province, Hefei 230036, P.R.China

Received:2020-10-22 Accepted:2021-06-04 Online:2022-04-01 Published:2021-06-04
About author:ZHU Lu, E-mail: 411143710@qq.com; JING Jing, E-mail: jingjing7209@163.com; Correspondence LING Ying-hui, Tel/Fax: +86-551-65785928, E-mail: lingyinghui@ahau.edu.cn * These authors contributed equally to this study.
Supported by:
This work was supported by the National Natural Science Foundation of China (31772566 and 31972629) and the Central Guidance on Local Science and Technology Development Fund of Anhui Province, China (202007d06020005).

摘要/Abstract

摘要：

MicroRNA对卵巢颗粒细胞的增殖、分化和分泌具有重要的调控作用，但miR-99a-5p在山羊卵巢颗粒细胞（GCs）中的作用尚不清楚。为探究miR-99a-5p在山羊GCs中的功能作用，我们从屠宰场收集了新鲜的山羊卵巢组织进行后续的荧光原位杂交和免疫组化试验；利用网站预测miR-99a-5p靶基因，通过双荧光素酶报告基因试验验证靶向关系；在分离培养的原代山羊卵巢颗粒细胞中过表达或敲低miR-99a-5p与其靶基因后，使用ELISA试剂盒检测细胞培养液中类固醇激素的含量。荧光原位杂交试验结果显示，miR-99a-5p在山羊卵巢颗粒细胞中表达，免疫组化结果显示其预测的靶基因FZD5也在颗粒细胞中表达；进一步的双荧光素酶报告基因试验表明FZD5是miR-99a-5p的一个靶基因（P<0.001），同时荧光定量和免疫印迹试验结果显示，过表达miR-99a-5p后，GCs中FZD5的mRNA和蛋白的表达量均会显著降低（P<0.05），反之亦然；利用过表达或敲低FZD5的慢病毒感染GCs后，可以显著改变细胞中FZD5的mRNA和蛋白的表达量，同时ELISA结果显示，过表达FZD5可显著提高细胞培养液中雌二醇和孕酮含量；过表达山羊卵巢GCs的miR-99a-5p后，细胞培养液中雌二醇和孕酮含量显著下降（P<0.05）。综上所述，miR-99a-5p抑制GCs中靶基因FZD5的表达以及雌二醇和孕酮的合成。因此，我们证实了山羊的FZD5是miR-99a-5p的一个靶基因；miR-99a-5p在体外抑制GCs雌二醇和孕酮的分泌，靶基因FZD5促进其分泌。本研究为山羊和其他动物卵泡发育的调控机制提供了重要的数据和新的见解。

Abstract: MicroRNA (miRNA) has vital regulatory effects on the proliferation, differentiation and secretion of ovarian granulosa cells, but the role of miR-99a-5p in goat ovarian granulosa cells (GCs) is unclear. Both miR-99a-5p and Frizzled-5 (FZD5) were found to be expressed in GCs in goat ovaries via fluorescence in situ hybridization and immunohistochemistry, respectively, and FZD5 was verified (P<0.001) as a target gene of miR-99a-5p by double luciferase reporter gene experiments. Furthermore, FZD5 mRNA and protein expression were both found to be regulated (P<0.05) by miR-99a-5p in GCs. Moreover, the overexpression of miR-99a-5p or knockdown of FZD5 suppressed (P<0.05) estradiol and progesterone secretion from the GCs, as determined by ELISA. In summary, miR-99a-5p inhibits target gene FZD5 expression and estradiol and progesterone synthesis in GCs. Our study thus provides seminal data and new insights into the regulatory mechanisms of follicular development in the goat and other animals.

Key words: miRNA , Frizzled-5 , follicular development , estradiol , progesterone

. [J]. Journal of Integrative Agriculture, 2022, 21(4): 1137-1145.

ZHU Lu, JING Jing, QIN Shuai-qi, LU Jia-ni, ZHU Cui-yun, ZHENG Qi, LIU Ya, FANG Fu-gui, LI Yun-sheng, ZHANG Yun-hai, LING Ying-hui. miR-99a-5p inhibits target gene FZD5 expression and steroid hormone secretion from goat ovarian granulosa cells[J]. Journal of Integrative Agriculture, 2022, 21(4): 1137-1145.

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miR-99a-5p inhibits target gene FZD5 expression and steroid hormone secretion from goat ovarian granulosa cells

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