CircACAP2对奶山羊乳腺上皮细胞凋亡及β-酪蛋白和甘油三酯合成的影响  

Effects of CircACAP2 on Apoptosis and Synthesis of β-casein and TG in Mammary Epithelial Cells of Dairy Goats(Capra hircus)

作　　者：江悦 朱俊儒 吴双虎 寇晓宇 安小鹏[1] 宋宇轩[1] JIANG Yue;ZHU Jun-Ru;WU Shuang-Hu;KOU Xiao-Yu;AN Xiao-Peng;SONG Yu-Xuan(College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China;Sanyuan County Animal Disease Prevention and Control Center,Sanyuan 713800,China;Chunhua County Agricultural and Rural Bureau,Xianyang 711200,China)

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]三原县动物疫病防控中心,三原713800 [3]淳化县农业农村局,咸阳711200

出　　处：《农业生物技术学报》2023年第9期1878-1889,共12页Journal of Agricultural Biotechnology

基　　金：陕西省重点研发计划(2020ZDLNY02-01);西北农林科技大学合水奶山羊试验示范基地(K4050422535);金昌肉羊试验示范基地(Z222021501)。

摘　　要：奶山羊(Capra hircus)品种选育的重要指标包括乳腺上皮细胞的数量和活性、泌乳量大小和乳成分。乳腺生长、发育及泌乳活动涉及复杂的调控过程。circACAP2(circular ArfGAP with coiled-coil,ankyrin repeat and PH domains 2)可以促进心肌细胞凋亡,同时在乳腺癌组织中高表达,加快乳腺癌进程,但其对奶山羊乳腺上皮细胞(goats mammary epithelial cells, GMECs)的调控作用尚不清楚。本研究构建circACAP2过表达和干扰载体,并转染GMECs,双荧光素酶活性分析结果显示,circACAP2能与miR-21靶向结合;qRT-PCR结果显示,circACAP2干扰使miR-21表达量显著提高。细胞增殖检测试剂盒CCK8(Cell Counting Kit-8)和5-乙炔基-2’-脱氧尿苷(5-ethynyl-2’-deoxyuridine, EdU)检测结果表明,circACAP2对GMECs增殖具有抑制作用;AnnexinV-FITC/PI细胞凋亡检测试剂盒和流式细胞仪检测结果显示,circACAP2对GMECs凋亡具有促进作用。ELISA试剂盒检测结果显示,circACAP2使GMECs的β-酪蛋白和甘油三酯分泌量降低。对ERK-Bax/Bcl-2凋亡信号通路和PI3K/mTOR/S6K泌乳信号通路相关分子进行Western blot检测,结果表明,circACAP2抑制pERK1/2和Bcl-2表达、促进Bax表达,同时PI3K、mTOR和S6K蛋白的磷酸化水平下降。上述研究结果表明,CircACAP2可能通过激活ERK-Bax/Bcl-2凋亡相关信号通路而促进GMECs凋亡,同时通过抑制泌乳相关的PI3K/mTOR/S6K信号通路而减少GMECs的β-酪蛋白和甘油三酯分泌。本研究为深入探讨关中奶山羊泌乳机理提供了基础资料。The number and activity of mammary epithelial cells,lactation volume and milk composition are very important indicators in the breed selection of dairy goats(Capra hircus).The growth,development and lactation of the mammary gland is an extremely complex process.circACAP2(circular ArfGAP with coiledcoil,ankyrin repeat and PH domains 2)can promote cardiomyocyte apoptosis while being highly expressed in breast cancer tissues,accelerating the progression of breast cancer.However,the regulatory effect of circACAP2 on goats mammary epithelial cells(GMECs)is not clear.In this study,circACAP2 overexpression and interference vector were constructed and transfected into GMECs,the results of double luciferase assay showed that circACAP2 could target binding with miR-21.qRT-PCR results showed that the interference with circACAP2 could significantly increase the expression of miR-21.The Cell proliferation test kit CCK8(Cell Counting Kit-8)and 5-ethynyl-2'-deoxyuridine(EdU)experiments showed that circACAP2 inhibited the proliferation of GMECs,and the AnnexinV-FITC/PI apoptosis assay kit and flow cytometry detection showed that circACAP2 promoted the apoptosis of GMECs.The detection of ELISA kit showed that circACAP2 reduced the secretion ofβ-casein and triglyceride(TG).The ERK-Bax/Bcl-2 apoptosis signaling pathway and PI3K/mTOR/S6K lactating signaling pathway were selected for Western blot detections.The results showed that circACAP2 inhibited the expression of pERK1/2 and Bcl-2 and promoted the expression of Bax.Meanwhile,the phosphorylation level of PI3K,mTOR,and S6K decreased.The above results indicate that CircACAP2 might affect the apoptosis and proliferation of GMECs through the apoptosis signaling pathway ERK-Bax/Bcl-2,and affect the secretion of β-casein and TG in GMECs through the lactating signaling pathway PI3K/mTOR/S6K.This study provides basic data for further study of the lactation mechanism of Guanzhong dairy goats.

关 键 词：CircACAP2(circular ArfGAP with coiled-coil ankyrin repeat and PH domains 2) MIR-21 奶山羊 细胞凋亡 Β-酪蛋白 甘油三酯(TG) 

分 类 号：S827[农业科学—畜牧学]