Please wait a minute...
Journal of Integrative Agriculture  2022, Vol. 21 Issue (4): 1161-1176    DOI: 10.1016/S2095-3119(21)63812-1
Special Issue: 动物科学合辑Animal Science
Animal Science · Veterinary Medicine Advanced Online Publication | Current Issue | Archive | Adv Search |
NH4Cl promotes apoptosis and inflammation in bovine mammary epithelial cells via the circ02771/miR-194b/TGIF1 axis
CHEN Zhi1, 2*, LIANG Yu-sheng3*, ZONG Wei-cheng4, GUO Jia-he1, 2, ZHOU Jing-peng1, 2, MAO Yong-jiang1, 2, JI De-jun1, 2, JIAO Pei-xin5, Juan J LOOR3, YANG Zhang-ping1, 2
1 College 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
5 College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      


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.
Keywords:  NH4Cl       circ02771        miR-194b        TGIF1        bovine mammary epithelial cells  
Received: 09 October 2020   Accepted: 16 August 2021
Fund: 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.
About author:  CHEN Zhi, E-mail:; Correspondence YANG Zhang-ping, Tel: +86-514-87979269, E-mail: * These authors contributed equally to this study.

Cite this article: 

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. 2022. NH4Cl promotes apoptosis and inflammation in bovine mammary epithelial cells via the circ02771/miR-194b/TGIF1 axis. Journal of Integrative Agriculture, 21(4): 1161-1176.

Bayat A R, Tapio I, Vilkki J, Shingfield K J, Leskinen H. 2018. Plant oil supplements reduce methane emissions and improve milk fatty acid composition in dairy cows fed grass silage-based diets without affecting milk yield. Journal of Dairy Science, 101, 1136–1151.
Bouwman A F, Lee D S, Asman W A H, Dentener F J, Van Der Hoek K W, Olivier J G J. 1997. A global high-resolution emission inventory for ammonia. Global Biogeochemical Cycles, 11, 561–587.
Chen D, Miao Z, Peng M, Xing H, Zhang H, Teng X. 2019. The co-expression of circRNA and mRNA in the thymuses of chickens exposed to ammonia. Ecotoxicology and Environmental Safety, 176, 146–152.
Chen W C, Park S H, Hoffman C, Philip C, Robinson L, West J, Grunig G. 2013. Right ventricular systolic pressure measurements in combination with harvest of lung and immune tissue samples in mice. Journal of Visualized Experiments, 16, e50023.
Chen Z, Chu S, Wang X, Sun Y, Xu T, Mao Y, Loor J J, Yang Z. 2019. MiR-16a regulates milk fat metabolism by targeting large tumor suppressor kinase 1 (LATS1) in bovine mammary epithelial cells. Journal of Agricultural and Food Chemistry, 67, 11167–11178.
Cui J, Han H, Piao J, Shi H, Zhou D, Gong X, Chang J. 2020. The construction of a novel biosensor based on the dual-enzyme cascade amplification-induced copper nanoparticles self-assembly for ultrasensitive detection of microRNA153. ACS Applied Materials & Interfaces, 30, 34130–34136. 
Elfsmark L, Agren L, Akfur C, Wigenstam E, Bergstrom U, Jonasson S. 2019. Comparisons of acute inflammatory responses of nose-only inhalation and intratracheal instillation of ammonia in rats. Inhalation Toxicology, 31, 107–118.
Feng Y, Wang Q, Shi C, Liu C, Zhang Z. 2019. Does circular rna exert significant effects in ovarian cancer? Critical Reviews in Eukaryotic Gene Expression, 29, 161–170.
Gerardo M L, Aljohani N H, Oatley-Radcliffe D L, Lovitt R W. 2015. Moving towards sustainable resources: Recovery and fractionation of nutrients from dairy manure digestate using membranes. Water Research, 80, 80–89.
Gilbreath K R, Bazer F W, Satterfield M C, Cleere J J, Wu G. 2020. Ruminal microbes of adult sheep do not degrade extracellular l-citrulline. Journal of Animal Science, 98, skaa164.
Hansen T B, Jensen T I, Clausen B H, Bramsen J B, Finsen B, Damgaard C K, Kjems J. 2013. Natural RNA circles function as efficient microRNA sponges. Nature, 495, 384–392.
Hosseini S M, Mesgaran M D, Vakili A R, Naserian A A, Khafipour E. 2019. Altering undigested neutral detergent fiber through additives applied in corn, whole barley crop, and alfalfa silages, and its effect on performance of lactating Holstein dairy cows. Asian–Australasian Journal of Animal Sciences, 32, 375–386.
Ismail F S, Faustmann T J, Corvace F, Tsvetanova A, Moinfar Z, Faustmann P M. 2021. Ammonia induced microglia activation was associated with limited effects on connexin 43 and aquaporin 4 expression in an astrocyte-microglia co-culture model. BMC Neuroscience, 22, 21.
Jiang Y, Ogunade I M, Arriola K G, Pech-Cervantes A A, Kim D H, Li X, Xue Y L, Vyas D, Adesogan A T. 2020. Short communication: Effects of a physiologically relevant concentration of aflatoxin B1 with or without sequestering agents on in vitro rumen fermentation of a dairy cow diet. Journal of Dairy Science, 103, 1559–1565.
Kou J, Liu G, Liu X, Li T, Wei Y, Sun Y, Wang T, Wang Y, Zheng X. 2020. Profiling and bioinformatics analysis of differentially expressed circrRNAs in spinal ligament tissues of patients with ankylosing spondylitis. BioMed Research International, 2020, 7165893.
La Y, He X, Zhang L, Di R, Wang X, Gan S, Zhang X, Zhang J, Hu W, Chu M. 2020. Comprehensive analysis of differentially expressed profiles of mRNA, lncRNA, and circRNA in the uterus of seasonal reproduction sheep. Genes (Basel), 11, 301.
Li L, Mao Y X, Zhao L N, Li L J, Wu J J, Zhao M J, Du W J, Yu L, Jiang P. 2019. p53 regulation of ammonia metabolism through urea cycle controls polyamine biosynthesis. Nature, 567, 253–256.
Liu J, Liu H, Zeng Q, Xu P, Liu M, Yang N. 2020. Circular RNA circ-MAT2B facilitates glycolysis and growth of gastric cancer through regulating the miR-515-5p/HIF-1 alpha axis. Cancer Cell International, 20, 171.
Ma Y, Zeng Z, Kong L, Chen Y, He P. 2019. Determination of N-carbamylglutamate in feeds and animal products by high performance liquid chromatography tandem mass spectrometry. Molecules, 24, 3172.
Mekuriaw S, Tsunekawa A, Ichinohe T, Tegegne F, Haregeweyn N, Kobayashi N, Tassew A, Mekuriaw Y, Walie M, Tsubo M, Okuro T, Meshesha D T, Meseret M, Sam L, Fievez V. 2020. Effect of feeding improved grass hays and eragrostis tef straw silage on milk yield, nitrogen utilization, and methane emission of lactating fogera dairy cows in ethiopia. Animals (Basel), 10, 1021.
Mulloy A L, Kari F W, Visek W J. 1982. Dietary arginine, insulin secretion, glucose tolerance and liver lipids during repletion of protein-depleted rats. Hormone and Metabolic Research, 14, 471–476.
Nomme J, Su Y, Konrad M, Lavie A. 2012. Structures of apo and product-bound human L-asparaginase: Insights into the mechanism of autoproteolysis and substrate hydrolysis. Biochemistry, 51, 6816–2686.
Ostolaza A, Blanco-Luquin I, Urdanoz-Casado A, Rubio I, Labarga A, Zandio B, Roldan M, Martinez-Cascales J, Mayor S, Herrera M, Aymerich N, Gallego J, Munoz R, Mendioroz M. 2020. Circular RNA expression profile in blood according to ischemic stroke etiology. Cell and Bioscience, 10, 34.
Pereira A B D, Whitehouse N L, Aragona K M, Schwab C S, Reis S F, Brito A F. 2017. Production and nitrogen utilization in lactating dairy cows fed ground field peas with or without ruminally protected lysine and methionine. Journal of Dairy Science, 100, 6239–6255.
Podralska M, Ciesielska S, Kluiver J, van den Berg A, Dzikiewicz-Krawczyk A, Slezak-Prochazka I. 2020. Non-coding RNAs in cancer radiosensitivity: MicroRNAs and lncRNAs as regulators of radiation-induced signaling pathways. Cancers (Basel), 12, 1662.
Sikorska H, Cianciara J, Wiercinska-Drapalo A. 2010. Physiological functions of L-ornithine and L-aspartate in the body and the efficacy of administration of L-ornithine–L-aspartate in conditions of relative deficiency. Review Pol Merkur Lekarski, 28, 490–495.
Taguchi A, Fahrmann J F, Hanash S M. 2020. A romising CPS1 inhibitor keeping ammonia from fueling cancer. Cell Chemical Biology, 27, 253–254.
Treacher R J, Little W, Collis K A, Stark A J. 1976. The influence of dietary protein intake on milk production and blood composition of high-yielding dairy cows. Journal of Dairy Research, 43, 357–369.
Vilarrasa-Nogue M, Teira-Esmatges M R, Gonzalez-Llinas E, Domingo-Olive F, Villar J M. 2020. Ammonia volatilisation from pig slurry and ANS with DMPP applied to Westerwold ryegrass (Lolium multiflorum Lam., cv. Trinova) under Mediterranean conditions. Science of the Total Environment, 724, 137918.
Visek W J. 1984. Ammonia: Its effects on biological systems, metabolic hormones, and reproduction. Journal of Dairy Science, 67, 481–498.
Wang D, Chen Z, Zhuang X, Luo J, Chen T, Xi Q, Zhang Y, Sun J. 2020. Identification of circRNA-associated-ceRNA networks involved in milk fat metabolism under heat stress. International Journal of Molecular Sciences, 21, 4162.
Wang F, Chen S, Jiang Y, Zhao Y, Sun L, Zheng B, Chen L, Liu Z, Zheng X, Yi K, Li C, Zhou X. 2018. Effects of ammonia on apoptosis and oxidative stress in bovine mammary epithelial cells. Mutagenesis, 33, 291–299.
Wang F G, Chen S X, Jiang Y W, Zhao Y, Sun L T, Zheng B B, Chen L, Liu Z, Zheng X, Yi K L, Li C J, Zhou X. 2018. Effects of ammonia on apoptosis and oxidative stress in bovine mammary epithelial cells. Mutagenesis, 33, 291–299.
Wang S, Wang W, Li X, Zhao X, Wang Y, Zhang H, Xu S. 2020. Cooperative application of transcriptomics and ceRNA hypothesis: LncRNA-107052630/miR-205a/G0S2 crosstalk is involved in ammonia-induced intestinal apoptotic injury in chicken. Journal of Hazardous Materials, 396, 122605.
Wang W, Shi Q, Wang S, Zhang H, Xu S. 2020. Ammonia regulates chicken tracheal cell necroptosis via the LncRNA-107053293/MiR-148a-3p/FAF1 axis. Journal of Hazardous Materials, 386, 121626.
Wei H, Liu D, Sun J, Mao Y, Zhao L, Zhu W, Xu G, Gao Z. 2019. Circular RNA circ_0008450 upregulates CXCL9 expression by targeting miR-577 to regulate cell proliferation and invasion in nasopharyngeal carcinoma. Experimental and Molecular Pathology, 110, 104288.
Xie Y, Li J, Li P, Li N, Zhang Y, Binang H, Zhao Y, Duan W, Chen Y, Wang Y, Du L, Wang C. 2020. RNA-Seq profiling of serum exosomal circular RNAs reveals Circ-PNN as a potential biomarker for human colorectal cancer. Frontiers in Oncology, 10, 982.
Yang D, Sun L, Li Z, Gao P. 2016. Noncoding RNAs in regulation of cancer metabolic reprogramming. Advances in Experimental Medicine and Biology, 927, 191–215.
Zhang C, Wu H, Wang Y, Zhu S, Liu J, Fang X, Chen H. 2016. Circular RNA of cattle casein genes are highly expressed in bovine mammary gland. Journal of Dairy Science, 99, 4750–4760.
Zhang J, Liu Y, Shi G. 2020. The circRNA-miRNA-mRNA regulatory network in systemic lupus erythematosus. Clinical Rheumatology, 40, 331–339.
Zhang L, Liu X, Che S, Cui J, Liu Y, An X, Cao B, Song Y. 2018. CircRNA-9119 regulates the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) by sponging miR-26a in the endometrial epithelial cells of dairy goat. Reproduction, Fertility and Development, 30, 1759–1769.
Zhao Z J, Shen J. 2017. Circular RNA participates in the carcinogenesis and the malignant behavior of cancer. RNA Biology, 14, 514–521.
Zheng Y, Chen C J, Lin Z Y, Li J X, Liu J, Lin F J, Zhou X. 2020. Circ_KATNAL1 regulates prostate cancer cell growth and invasiveness through the miR-145-3p/WISP1 pathway. Biochemistry and Cell Biology, 98, 396–404.
Zhu Q, Zhang Q, Gu M, Zhang K, Xia T, Zhang S, Chen W, Yin H, Yao H, Fan Y, Pan S, Xie H, Liu H, Cheng T, Zhang P, Zhang T, You B, You Y. 2020. MIR106A-5p upregulation suppresses autophagy and accelerates malignant phenotype in nasopharyngeal carcinoma. Autophagy, 17, 1667–1683.

No related articles found!
No Suggested Reading articles found!