Effects of Epidermal Growth Factor (EGF) on Autophagy and Endocytosis-related Protein Expression in Mouse (Mus musculus) Cumulus Cells
MA Rui1,2, WANG Meng2, LI Qin2, HE Hong-Hong2, WANG Jing-Lei2, CUI Yan2, YU Si-Jiu2, PAN Yang-Yang2,*
1 College of Life Science and Technology, Gansu Agricultural University, Lanzhou 730070, China; 2 College of Veterinary Medicine/Technology and Research Center of Gansu Province for Embryonic Engineering of Bovine and Sheep & Goat, Gansu Agricultural University, Lanzhou 730070, China;
Abstract：Autophagy and endocytosis are important life activities in the process of cell growth and development, which can regulate the growth state of cells to cope with the threat of harsh environment. To investigate the effect of epidermal growth factor (EGF) on autophagy and endocytosis of mouse (Mus musculus) cumulus cells, the concentrations 0, 50, 100, 150, 200 ng/mL of EGF and appropriate concentration (40 μg/mL) epidermal growth factor receptor (EGFR) inhibitor Gefitinib were applied to mouse cumulus cells, and cellular total RNA and total protein were extracted. The relative expression levels of autophagy related gene 5 (Atg5), Bcl-2 homologous domain protein (Beclin1), microtubule-associated protein light chain 3 (LC3), caveolin 1 (Cav1) and caveolin 2 (Cav2) mRNA at various concentrations were determined by qRT-PCR. The relative expression levels of Atg5, Beclin1, LC3, Cav1 and Cav2 proteins in every concentration were determined by Western-blot. The protein expressions were localized by immunofluorescence technique. The results showed that Atg5, Beclin1, LC3, Cav1 and Cav2 proteins were expressed in the normally growing mouse cumulus cells, and the main expression site was cytoplasm. After EGF treatments, gene expression levels of Atg5, LC3, and Cav1 increased significantly and were highest at 150 ng / mL (P<0.05), while the relative expression levels of Beclin1 and Cav2 decreased significantly and got lowest at 150 and 200 ng/mL (P<0.05). Protein expression levels of Atg5, Beclin1, and LC3 all increased significantly (P<0.05) and were highest at 150~200 ng/mL (P<0.05). In addition, LC3Ⅰ and LC3Ⅱ proteins were highest at 200 ng/mL and LC3Ⅱ/LC3Ⅰ was highest at 100 ng / mL. And Cav1 and Cav2 proteins were significantly increased at 50 ng/mL (P<0.05), while they were significantly decreased at 200 ng/mL (P<0.05). After inhibiting EGFR, the relative expression levels of Atg5, LC3, and Cav1 genes were significantly increased, while the expression levels of Beclin1 and Cav2 genes were significantly reduced (P<0.05). Protein expression levels of Atg5, Beclin1, LC3, LC3Ⅱ/LC3Ⅰ, and Cav1 were significantly increased (P<0.05, P<0.01), while the expression of Cav2 protein was significantly decreased (P<0.01). The results showed that EGF factor played an important role in the autophagy and endocytosis of mouse cumulus cells. The levels of autophagy and endocytosis were dose-dependent with the concentration of EGF. This study provides theoretical basis for further study of the regulation mechanism of EGF factor on autophagy and endocytosis of mouse cumulus cells.
马睿, 王萌, 李秦, 何翃闳, 王靖雷, 崔燕, 余四九, 潘阳阳. 表皮生长因子(EGF)对小鼠卵丘细胞自噬及胞吞相关蛋白表达的影响[J]. 农业生物技术学报, 2020, 28(1): 119-128.
MA Rui, WANG Meng, LI Qin, HE Hong-Hong, WANG Jing-Lei, CUI Yan, YU Si-Jiu, PAN Yang-Yang. Effects of Epidermal Growth Factor (EGF) on Autophagy and Endocytosis-related Protein Expression in Mouse (Mus musculus) Cumulus Cells. 农业生物技术学报, 2020, 28(1): 119-128.
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