NH4Cl promotes apoptosis and inflammation in bovine mammary epithelial cells via the circ02771/miR-194b/TGIF1 axis

doi:10.1016/S2095-3119(21)63812-1

Journal of Integrative Agriculture ›› 2022, Vol. 21 ›› Issue (4): 1161-1176.DOI: 10.1016/S2095-3119(21)63812-1

收稿日期:2020-10-09 接受日期:2021-08-16 出版日期:2022-04-01 发布日期:2021-08-16

NH₄Cl promotes apoptosis and inflammation in bovine mammary epithelial cells via the circ02771/miR-194b/TGIF1 axis

CHEN Zhi^{1, 2*}, LIANG Yu-sheng^3*, ZONG Wei-cheng⁴, GUO Jia-he^{1, 2}, ZHOU Jing-peng^{1, 2}, MAO Yong-jiang^{1, 2}, JI De-jun^{1, 2}, JIAO Pei-xin⁵, Juan J LOOR³, YANG Zhang-ping^{1, 2}

¹College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, P.R.China
² Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education, Yangzhou University, Yangzhou 225009, P.R.China
³ Mammalian Nutrition Physiology Genomics, Department of Animal Sciences and Division of Nutritional Sciences, University of Illinois, Urbana, IL 61801, USA
⁴ College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, P.R.China
⁵College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, P.R.China

Received:2020-10-09 Accepted:2021-08-16 Online:2022-04-01 Published:2021-08-16
About author:CHEN Zhi, E-mail: zhichen@yzu.edu.cn; Correspondence YANG Zhang-ping, Tel: +86-514-87979269, E-mail: yzp@yzu.edu.cn * These authors contributed equally to this study.
Supported by:
This research was supported by the Independent Innovation in Jiangsu Province of China (CX (21) 3119), the National Natural Science Foundation of China (31802035, 31702095 and 31872324), the Jiangsu Natural Science Fund (BK20181221), and Yangzhou Liangde Antibody BioTech., China.

摘要/Abstract

摘要：

奶畜动物体内中含过量的氨不但会降低其自身健康而且还会生产出对人体有害的食品。为了开发有效的预防和治疗方法，有必要对奶牛体内含有过量NH3的分子机制展开一系列研究。本研究使用奶牛乳腺上皮细胞为研究对象，通过高通量测序，实时定量PCR，Westernblot分析，荧光素酶报告实验，流式细胞分析，透射电镜，H&E染色和免疫组织化学等方法展开实验。利用NH₄Cl处理奶牛乳腺上皮细胞，构建NH₄Cl诱导的炎症模型和NH₄Cl处理的奶牛乳腺上皮细胞circRNAs表达谱，以空白组为对照组，NH₄Cl处理的奶牛乳腺上皮细胞为实验组，获得28个CircRNA差异大于2倍，P值小于0.05，其中17个上调，11个下调。circ02771显示出最显著的差异，倍数变化为4.12。通过绘制circRNA-miRNA靶点的相互作用网络图，发现共有255对circRNA-miRNA，包括circRNA-02771/miR-194b。序列分析表明，circ02771源于14号染色体上的26360485-26402413，在circ02771中存在一个amiR-194b结合位点。实验结果也表明，与野生型载体共转染后，miR-194b的荧光素酶活性显著降低（P<0.05），但与突变型载体共转染后，荧光素酶活性无显著差异（P<0.01）。circRNA-02771的过度表达显著降低（P<0.01）miR-194b的表达。证明circRNA-02771/miR-194b调控关系是成立的。实验分析发现miR-194b是与TGIF1的3'-UTR结合的miRNA之一。TGIF1的表达被miR-194b模拟物下调（P<0.05）。荧光素酶报告分析的结果表明，miR-194b的过度表达降低了野生型报告基因3'-UTR中的荧光素酶活性（P<0.01），但突变报告基因中的荧光素酶活性没有显著改变。结果证明miR-194b特异性的靶向TGIF1。流式细胞术结果显示circ02771和TGIF1显著增加奶牛乳腺上皮细胞的凋亡。EdU实验显示circ02771和TGIF1能够使细胞中的荧光强度降低。表达水平检测显示circ02771和TGIF1显著增强（P<0.01）PGLYRP1、和PTX3的表达。这些研究证明circ02771和TGIF1促进奶牛乳腺上皮细胞凋亡和炎症。我们的数据表明，NH₄Cl通过circ02771/miR-194b/TGIF1轴导致奶牛乳腺上皮细胞凋亡和炎症。

Abstract: Excess ammonia (NH3) in the circulation of dairy animals can reduce animal health and the quality of products for human consumption. To develop effective prevention and treatment methods, it is essential to examine the molecular mechanisms through which excess NH3 may affect the mammary gland. The present study used bovine mammary epithelial cells (BMECs) to evaluate the effects of exogenous NH₄Cl on the abundance of circular RNAs (circRNAs) using high-throughput sequencing. Among the identified circRNAs, circ02771 was the most significantly upregulated by exogenous NH₄Cl (P<0.05), with a fold change of 4.12. The results of the apoptosis and proliferation assays, transmission electron microscopy, H&E staining, and immunohistochemistry revealed that circ02771 increased apoptosis and inflammation. A double luciferase reporter assay revealed that circ02771 targeted miR-194b, and the overexpression of circ02771 (pcDNA-circ02771) reduced (P<0.05) the expression of miR-194b and led to apoptosis and inflammation. Circ02771 also enhanced the expression of transforming growth factor beta-induced factor homeobox 1 (TGIF1), which is a target gene of miR-194b. Overall, this study suggests that the circ02771/miR-194b/TGIF1 axis plays a role in mediating the effects of NH₄Cl on BMECs. Therefore, this axis provides a novel target to help control hazards within the mammary gland from high circulating NH₄Cl levels.

Key words: NH₄Cl , circ02771 , miR-194b , TGIF1 , bovine mammary epithelial cells

. [J]. Journal of Integrative Agriculture, 2022, 21(4): 1161-1176.

CHEN Zhi, LIANG Yu-sheng, ZONG Wei-cheng, GUO Jia-he, ZHOU Jing-peng, MAO Yong-jiang, JI De-jun, JIAO Pei-xin, Juan J LOOR, YANG Zhang-ping. NH₄Cl promotes apoptosis and inflammation in bovine mammary epithelial cells via the circ02771/miR-194b/TGIF1 axis[J]. Journal of Integrative Agriculture, 2022, 21(4): 1161-1176.

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NH₄Cl promotes apoptosis and inflammation in bovine mammary epithelial cells via the circ02771/miR-194b/TGIF1 axis

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