PDCD4对奶山羊乳腺上皮细胞的凋亡及β-酪蛋白和TG合成的影响

doi:10.11843/j.issn.0366-6964.2023.04.008

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (4): 1429-1440.doi: 10.11843/j.issn.0366-6964.2023.04.008

PDCD4对奶山羊乳腺上皮细胞的凋亡及β-酪蛋白和TG合成的影响

朱俊儒¹, 韦唯², 裴党帅³, 张芬鹊⁴, 段瑜⁵, 郭永峰⁶, 江悦¹, 夏树立⁷, 韩静⁷, 侯金星⁸, 安小鹏^1*

1. 西北农林科技大学动物科技学院, 杨凌 712100;
2. 渭南市动物疫病预防控制中心, 渭南 714000;
3. 渭南市动物卫生工作站, 渭南 714000;
4. 渭南市农产品质量安全检验检测中心, 渭南 714000;
5. 渭南市农业技术推广中心, 渭南 714000;
6. 黄骅市农业农村局, 黄骅 061100;
7. 天津市农业科学院畜牧兽医研究所, 天津 300112;
8. 杨凌职业技术学院, 杨凌 712100

收稿日期:2022-07-18 出版日期:2023-04-23 发布日期:2023-04-27
通讯作者: 安小鹏,主要从事动物遗传育种与繁殖研究,E-mail:anxiaopengdky@163.com
作者简介:朱俊儒(1998-),男,河北沧州人,硕士生,主要从事动物遗传育种与繁殖研究,E-mail:zjr18710872378@163.com
基金资助:
陕西省重点研发计划（2021NY-001；2020ZDLNY02-01）；杨凌职业技术学院科学研究基金（A2017003）；杨凌职业技术学院自然科学研究基金（A2019067）；优质肉羊种质资源的引进、评价和利用（19ZXZYSN00010）

Effects of PDCD4 on the Apoptosis of Dairy Goat Mammary Epithelial Cells and the Synthesis of β-casein and TG

ZHU Junru¹, WEI Wei², PEI Dangshuai³, ZHANG Fenque⁴, DUAN Yu⁵, GUO Yongfeng⁶, JIANG Yue¹, XIA Shuli⁷, HAN Jing⁷, HOU Jinxing⁸, AN Xiaopeng^1*

1. College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China;
2. Weinan Animal Disease Prevention and Control Center, Weinan 714000, China;
3. Weinan Animal Health Workstation, Weinan 714000, China;
4. Weinan Agricultural Product Quality and Safety Inspection and Testing Center, Weinan 714000, China;
5. Weinan Agricultural Technology Promotion Center, Weinan 714000, China;
6. Huanghua Municipal Bureau of Agriculture and Rural Affairs, Huanghua 061100, China;
7. Institute of Animal Husbandry and Veterinary Medicine, Tianjin Academy of Agriculture Sciences, Tianjin 300112, China;
8. Yangling Vocational Technical College, Yangling 712100, China

Received:2022-07-18 Online:2023-04-23 Published:2023-04-27

摘要/Abstract

摘要： 旨在探究奶山羊程序性细胞死亡因子4（programmed cell death 4，PDCD4）对乳腺上皮细胞（goat mammary epithelial cells，GMECs）凋亡及乳成分的调控作用。本研究通过双荧光素酶报告试验验证miR-21-5p与PDCD4的靶向关系，通过RT-qPCR、Western blot试验检测miR-21-5p对PDCD4的调控作用；将PDCD4干扰小RNA （si-PDCD4）与PDCD4过表达载体（pc3.1-PDCD4）转染进GMECs以探究PDCD4对GMECs的影响，通过EdU、CCK-8试验检测转染后对GMECs增殖的影响，利用流式细胞术检测转染后对GMECs凋亡的影响；利用ELISA试剂盒检测转染后GMECs中β-酪蛋白和甘油三酯（TG）的分泌情况；最后通过Western blot试验检测转染后GMECs中PI3K、mTOR、S6K、ERK、Bcl-2、Bax等蛋白的表达。双荧光素酶报告试验结果表明，PDCD4与miR-21-5p靶向结合；EdU、CCK-8及流式细胞术试验结果表明，过表达PDCD4后GMECs活力显著降低（P<0.01），即PDCD4抑制GMECs增殖并促进GMECs凋亡（P<0.01），干扰PDCD4的表达后GMECs活力显著提高（P<0.01），GMECs的凋亡水平显著降低（P<0.01）；ELISA试验结果表明，过表达PDCD4后GMECs中β-酪蛋白和TG的分泌量显著降低（P<0.01），降低PDCD4的表达后GMECs中β-酪蛋白和TG的分泌量显著上升（P<0.01）；Western blot试验结果表明，与对照组相比，过表达PDCD4后磷酸化的PI3K、mTOR、S6K、ERK蛋白表达显著降低（P<0.05），Bax蛋白表达被显著上调（P<0.01），Bcl-2蛋白表达水平被显著下调（P<0.01）；降低PDCD4的表达后，PI3K、mTOR、S6K、ERK的磷酸化蛋白表达显著提高（P<0.05），Bax的表达则显著下降（P<0.05），同时显著上调了Bcl-2的表达（P<0.05）。本研究结果表明，PDCD4通过激活ERK/Bcl-2/Bax信号通路促进奶山羊乳腺上皮细胞的凋亡，并通过抑制PI3K/mTOR/S6K信号通路降低奶山羊乳腺上皮细胞中β-酪蛋白和TG的分泌，miR-21-5p在GMECs中与PDCD4靶向结合并调控其表达。

关键词: PDCD4, miR-21-5p, 奶山羊, 凋亡, β-酪蛋白, TG

Abstract: The aim of this study was to investigate the regulatory effects of programmed cell death 4 (PDCD4) on the apoptosis of mammary epithelial cells (GMECs) and milk composition in goat. In this study, the dual luciferase reporter assay was used to verify the targeting relationship between miR-21-5p and PDCD4, and the regulatory effects of miR-21-5p on PDCD4 was detected by RT-qPCR and Western blot. The PDCD4 small interfering RNA (si-PDCD4) and PDCD4 overexpression vector (pc3.1-PDCD4) were transfected into GMECs to explore the effect of PDCD4. EdU and CCK-8 tests were used to detect the effect of PDCD4 on the proliferation of GMECs, flow cytometry was chosen to detect its effect on apoptosis of GMECs. After transfection, the ELISA kit was used to detect the secretion of β-casein and triglyceride (TG) in GMECs. Finally, the Western blot tests was used to detect the protein expression of PI3K, mTOR, S6K, ERK, Bcl-2, Bax in GMECs. The results of the dual luciferase reporter assay showed that PDCD4 was one of the target genes of miR-21-5p. The results of the EdU, CCK-8 and flow cytometry tests showed that the activity of GMECs was significantly decreased after overexpression of PDCD4 (P<0.01), indicated that PDCD4 inhibited the proliferation of GMECs and promoted the apoptosis of GMECs (P<0.01); the activity of GMECs was significantly increased after interfering PDCD4 (P<0.01), the pro-apoptotic level of GMECs was significantly reduced (P<0.01). The results of the ELISA kit test showed that the secretion of β-casein and TG in GMECs was significantly decreased after overexpression of PDCD4 (P<0.01), and they were significantly increased after interfering PDCD4 (P<0.01). The results of the Western blot test showed that, compared with the control group, the protein expressions of phosphorylated PI3K, mTOR, S6K and ERK were significantly decreased after overexpression PDCD4 (P<0.05), the protein expression of Bax was significantly up-regulated (P<0.01), and the protein expression of Bcl-2 was significantly down-regulated (P<0.01). After interfering PDCD4, the protein expression of phosphorylated PI3K, mTOR, S6K and ERK were significantly increased (P<0.05), the protein expression of Bax was significantly decreased (P<0.05), and the expression of Bcl-2 was significantly up-regulated (P<0.05). This study indicates that PDCD4 promotes the apoptosis of dairy goat mammary epithelial cells by activating the ERK/Bcl-2/Bax signaling pathway, and decreases the secretion of β-casein and TG in dairy goat mammary epithelial cells by inhibiting PI3K/mTOR/S6K signaling pathway. And miR-21-5p targets PDCD4 and can regulate its expression in GMECs.

Key words: PDCD4, miR-21-5p, milk goat, apoptosis, beta-casein, TG

中图分类号:

S827.2

朱俊儒, 韦唯, 裴党帅, 张芬鹊, 段瑜, 郭永峰, 江悦, 夏树立, 韩静, 侯金星, 安小鹏. PDCD4对奶山羊乳腺上皮细胞的凋亡及β-酪蛋白和TG合成的影响[J]. 畜牧兽医学报, 2023, 54(4): 1429-1440.

ZHU Junru, WEI Wei, PEI Dangshuai, ZHANG Fenque, DUAN Yu, GUO Yongfeng, JIANG Yue, XIA Shuli, HAN Jing, HOU Jinxing, AN Xiaopeng. Effects of PDCD4 on the Apoptosis of Dairy Goat Mammary Epithelial Cells and the Synthesis of β-casein and TG[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1429-1440.

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PDCD4对奶山羊乳腺上皮细胞的凋亡及β-酪蛋白和TG合成的影响

Effects of PDCD4 on the Apoptosis of Dairy Goat Mammary Epithelial Cells and the Synthesis of β-casein and TG

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