miR-144-5p靶向WNT5a对山羊卵巢颗粒细胞增殖、凋亡的影响

doi:10.11843/j.issn.0366-6964.2023.06.021

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (6): 2421-2435.doi: 10.11843/j.issn.0366-6964.2023.06.021

miR-144-5p靶向WNT5a对山羊卵巢颗粒细胞增殖、凋亡的影响

刘杰^1,2, 许香萍^1,2, 邓铭^1,3,4, 邹娴^1,2, 江声伟^1,2, 刘德武^1,3,4, 柳广斌^1,3,4, 孙宝丽^1,3,4, 郭勇庆^1,3,4, 李耀坤^1,3,4*

1. 华南农业大学动物科学学院, 广州 510642;
2. 广东省农业科学院动物科学研究所, 畜禽育种国家重点实验室, 广东省畜禽育种与营养研究重点实验室, 广州 510640;
3. 华南农业大学畜禽育种国家地方联合工程研究中心, 广州 510642;
4. 华南农业大学, 广东省农业动物基因组学与分子育种重点实验室, 广州 510642

收稿日期:2022-08-30 出版日期:2023-06-23 发布日期:2023-06-16
通讯作者: 李耀坤,主要从事动物繁殖与生产研究,E-mail:ykli@scau.edu.cn
作者简介:刘杰(1998-),男,山东临沂人,硕士生,主要从事动物遗传育种与繁殖研究,E-mail:1968286470@qq.com;许香萍(1997-),女,广东潮州人,硕士生,主要从事动物繁殖与生产研究,E-mail:99502921616@qq.com
基金资助:
广东省现代农业产业技术体系项目(2021KJ127);广东省科技特派员项目(KTP20210344)

Effect of miR-144-5p Targeting WNT5a on the Proliferation and Apoptosis of Goat Ovarian Granulosa Cells

LIU Jie^1,2, XU Xiangping^1,2, DENG Ming^1,3,4, ZOU Xian^1,2, JIANG Shengwei^1,2, LIU Dewu^1,3,4, LIU Guangbin^1,3,4, SUN Baoli^1,3,4, GUO Yongqing^1,3,4, LI Yaokun^1,3,4*

1. College of Animal Science, South China Agricultural University, Guangzhou 510642, China;
2. Guangdong Key Laboratory of Animal Breeding and Nutrition, State Key Laboratory of Livestock and Poultry Breeding, Institute of Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China;
3. National Engineering Research Center for Livestock and Poultry Breeding, South China Agricultural University, Guangzhou 510642, China;
4. Guangdong Key Laboratory of Agricultural Animal Genomics and Molecular Breeding, South China Agricultural University, Guangzhou 510642, China

Received:2022-08-30 Online:2023-06-23 Published:2023-06-16

摘要/Abstract

摘要： 旨在探究miR-144-5p靶向WNT5a对山羊颗粒细胞增殖和凋亡的影响。本研究选用2岁健康雷州母山羊的卵巢颗粒细胞来过表达和抑制表达miR-144-5p及WNT5a,通过荧光定量PCR(real-time qPCR, RT-qPCR)、Western blot、双荧光素酶报告基因、CCK-8等技术来探究miR-144-5p与WNT5a对颗粒细胞增殖和凋亡的影响。结果表明,miR-144-5p在大卵泡颗粒细胞中的表达量极显著高于小卵泡颗粒细胞(P<0.01),WNT5a在大卵泡颗粒细胞中的表达量极显著低于小卵泡颗粒细胞(P<0.01);CCK-8检测发现,miR-144-5p抑制了GCs增殖,过表达miR-144-5p后,抑凋亡基因BCL-2的mRNA和蛋白表达极显著下调(P<0.01),促凋亡基因BAX的mRNA和蛋白表达极显著上调(P<0.01);Wnt/β-Catenin通路基因CTNNB1 mRNA和β-Catenin蛋白水平极显著降低(P<0.01);另外,miR-144-5p能靶向WNT5a抑制其表达。同时,CCK-8检测结果表明,WNT5a可促进GCs增殖,过表达WNT5a后,抑凋亡基因BCL-2的mRNA和蛋白水平极显著上调(P<0.01),促凋亡基因BAX的mRNA显著下调(P<0.05)、蛋白水平极显著下调(P<0.01),Wnt/β-Catenin通路CTNNB1 mRNA和β-Catenin蛋白水平极显著提高(P<0.01)。综上所述,在GCs中,miR-144-5p靶向负调控WNT5a的表达,调控Wnt/β-Catenin通路,进而上调BAX/BCL-2的比值来抑制颗粒细胞增殖并促进其凋亡,这可能对山羊的排卵数和产羔数产生一定的影响。

关键词: 颗粒细胞, miR-144-5p, WNT5a, 山羊, 繁殖

Abstract: The study aimed to investigate the effect of miR-144-5p targeting WNT5a on proliferation and apoptosis of goat granulosa cells. Ovarian granulosa cells from 2-year-old healthy Leizhou female goats were selected to overexpress and repress miR-144-5p and WNT5a, and the effects of miR-144-5p and WNT5a on granulosa cell proliferation and apoptosis were investigated by fluorescent quantitative PCR (real-time qPCR, RT-qPCR), Western blot, dual luciferase reporter gene, CCK-8 and other techniques. The results showed that the expression of miR-144-5p was significantly higher in large follicular granulosa cells than that in small follicular granulosa cells (P<0.01), and the expression of WNT5a was significantly lower in large follicular granulosa cells than that in small follicular granulosa cells (P<0.01); CCK-8 assay revealed that miR-144-5p inhibited the proliferation of GCs, and overexpression of miR-144-5p resulted in significant downregulation of mRNA and protein expression of the apoptosis suppressor gene BCL-2 (P<0.01) and significant upregulation of mRNA and protein expression of the pro-apoptotic gene BAX (P<0.01); Wnt/β-Catenin pathway gene CTNNB1 mRNA and β-Catenin protein levels were highly significantly reduced (P<0.01); In addition, miR-144-5p could target WNT5a to inhibit its expression. Meanwhile, CCK-8 assay results showed that WNT5a promoted the proliferation of GCs, and overexpression of WNT5a significantly upregulated the mRNA and protein levels of the BCL-2 (P<0.01), significantly downregulated the mRNA level (P<0.05) and protein level (P<0.01) of BAX, and the Wnt/β-Catenin pathway gene CTNNB1 mRNA and β-Catenin protein levels were significantly increased (P<0.01). In summary, in GCs, miR-144-5p targets and negatively regulates WNT5a expression, regulates the Wnt/β-Catenin pathway, and then upregulates the BAX/BCL-2 ratio to inhibit granulosa cell proliferation and promote apoptosis, which may have an effect on ovulation and lambing numbers in goats.

Key words: granulosa cells, miR-144-5p, WNT5a, goat, reproduction

中图分类号:

S827.3

刘杰, 许香萍, 邓铭, 邹娴, 江声伟, 刘德武, 柳广斌, 孙宝丽, 郭勇庆, 李耀坤. miR-144-5p靶向WNT5a对山羊卵巢颗粒细胞增殖、凋亡的影响[J]. 畜牧兽医学报, 2023, 54(6): 2421-2435.

LIU Jie, XU Xiangping, DENG Ming, ZOU Xian, JIANG Shengwei, LIU Dewu, LIU Guangbin, SUN Baoli, GUO Yongqing, LI Yaokun. Effect of miR-144-5p Targeting WNT5a on the Proliferation and Apoptosis of Goat Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(6): 2421-2435.

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miR-144-5p靶向WNT5a对山羊卵巢颗粒细胞增殖、凋亡的影响

Effect of miR-144-5p Targeting WNT5a on the Proliferation and Apoptosis of Goat Ovarian Granulosa Cells

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