SNAT2介导蛋氨酸对牛乳腺上皮细胞增殖与自噬的调节作用

doi:10.16431/j.cnki.1671-7236.2019.09.003

摘要/Abstract

摘要： 钠离子依赖性中性氨基酸转运体2（SNAT2）是一种氨基酸转运蛋白，可转运中性氨基酸，广泛分布于多种细胞中。氨基酸既可作为蛋白质合成的底物，也是调节细胞新陈代谢的关键信号分子，但SNAT2是否介导氨基酸调节BMECs增殖和自噬尚未见报道。本研究利用CASY细胞计数和Western blotting技术检测SNAT2过表达和siRNA干扰后牛乳腺上皮细胞（BMECs）增殖情况以及SNAT2对自噬标志蛋白LC3-Ⅰ/Ⅱ表达量的影响，并利用免疫荧光检测细胞自噬斑点（LC3-Ⅱ）变化。结果显示，SNAT2过表达时，p-PI3K、p-mTOR和Cyclin D1表达量增加，反之，p-PI3K、p-mTOR和Cyclin D1表达量下降。SNAT2抑制时，LC3-Ⅱ表达量增加，免疫荧光检测自噬斑点增多。添加自噬增强剂海藻糖（trehalose，Tre）和蛋氨酸（methionine，Met）后，与单一添加Tre组相比，Met+Tre组p-mTOR表达量增加，LC3-Ⅱ表达量降低，胞浆内绿色自噬斑点减少；添加Tre和Met并抑制SNAT2时，p-mTOR表达量下降，LC3-Ⅱ表达量增多，胞浆内绿色自噬斑点增加。以上结果表明，SNAT2可介导Met通过调控PI3K-mTOR/Cyclin D1信号通路调节BMECs的增殖与自噬。

关键词: 钠离子依赖性中性氨基酸转运体2(SNAT2); 牛乳腺上皮细胞(BMECs); mTOR; 增殖; 自噬

Abstract: The sodium-dependent neutral amino acid transporter 2 (SNAT2) is an amino acid transporter which can transport neutral amino acids and is widely distributed in various cells.Amino acids is not only substrates for protein synthesis,but also key signal molecules regulating cell metabolism.However,it is little known whether SNAT2 mediates the regulation of amino acids on autophagy.In this study,CASY cell count and Western blotting were used to determine the proliferation of BMECs after SNAT2 overexpression and siRNA interference.The effects of SNAT2 on the expression of autophagic biomarker LC3-Ⅱ was detected by Western blotting and the change of autophagic spots (LC3-Ⅱ) in cells was determined by immunofluorescence.The results showed that SNAT2 overexpression increased the expression levels of p-PI3K,p-mTOR and cyclin D1,whereas SNAT2 knockdown had the opposite effects.Furthermore,the expression of LC3-Ⅱ and the autophagy spots were both increased in SNAT2-downregulated cells.The p-mTOR expression was increased and LC3-Ⅱ was decreased in cells treated with the autophagy enhancer trehalose dihydrate (Tre) together with methionine (Met) compared with the control (cells treated with Tre).The expression of p-mTOR was decreased and LC3-Ⅱ was increased in cells treated with Tre together with Met addition and transfected with a SNAT2 siRNA compared with the control (cells treated with Tre and Met).In conclusion,these data demonstrated that SNAT2 mediated the regulation of Met on proliferation of and autophagy in BMECs via PI3K-mTOR/Cyclin D1 signaling pathway.

Key words: SNAT2; BMECs; mTOR; proliferation; autophagy

中图分类号:

Q291

祁昊, 孟春雨, 高学军, 甄贞. SNAT2介导蛋氨酸对牛乳腺上皮细胞增殖与自噬的调节作用[J]. 中国畜牧兽医, 2019, 46(9): 2516-2525.

QI Hao, MENG Chunyu, GAO Xuejun, ZHEN Zhen. SNAT2: A Key Mediator for Methionine to Regulate Proliferation and Autophagy in Bovine Mammary Epithelial Cells[J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(9): 2516-2525.

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