DNA甲基化通过调控细胞自噬影响家蚕翅的发育

目的]本研究旨在探索DNA甲基化是否通过调控细胞自噬进而影响家蚕Bombyx mori翅的发育.方法]分别用1和2 μg DNA甲基化特异性抑制剂5-aza-dC处理家蚕卵巢Bm12细胞和家蚕预蛹,荧光显微镜下观察Bm12细胞的数量,利用dot blot检测Bm12细胞中DNA甲基化水平,利用溶酶体染色检测细胞自噬...

DNA methylation affects wing development of Bombyx mori through regulating autophagy

【Aim】This study aims to investigate whether DNA methylation affects wing development of Bombyx mori through regulating autophagy. 【Methods】B. mori ovarian Bm12 cells and B. mori prepupae were treated with 5 aza dC, a DNA methylation inhibitor, at the doses of 1 and 2 μg, respectively, the number of Bm12 cells was observed under fluorescence microscopy, the methylation level in Bm12 cells was detected by dot blotting, the autophagy intensity was detected by lysosome staining, the expression levels of autophagy related protein (Atg) genes were detected by RT-qPCR, and the typing of the autophagy marker LC3 protein in Bm12 cells was detected by Western blotting and immunohistochemistry. Meanwhile, the wing phenotype of adult was observed, and the abnormal wing rate and wing area were calculated. After B. mori prepupae were treated with the autophagy activator SMER28 (2 μg), and using autophagy inhibitor Spautin-1 (2 μg) to rescue 5-aza-dC-treated prepupae, the autophagy intensity in the wing cells was detected by lysosome staining, the wing phenotype was observed, and the abnormal wing rate and wing area were calculated. 【Results】Treatment with 1 μg of 5-aza-dC inhibited the growth and decreased the methylation level of Bm12 cells, increased the autophagy level in Bm12 cells at 12, 24 and 48 h after treatment, and up-regulated the expression of Atg genes in Bm12 cells at 48 h after treatment. Injection of 2 μg of 5-aza-dC into B. mori prepupae increased the autophagy level, up-regulated the expression of Atg genes in adult wing cells, and resulted in a large proportion of deformed wings at 24, 48 and 72 h after treatment, with the increased abnormal wing rate (increased by 72.62%) and the decreased wing area (decreased by 66%) of adult. Injection of 2 μg of SMER28 into B. mori prepupae increased the autophagy level in the adult wing cells and resulted in a large proportion of deformed wings at 24, 48 and 72 h after treatment, with the increased abnormal wing rate (increased by 75.13%) and the decreased wing area (decreased by 48.79%) of adult. In addition, rescue experiment using Spautin-1 to rescue the 5-aza-dC-treated prepupae revealed that the autophagy inhibition could alleviate the effect of DNA demethylation on the adult wing development. 【Conclusion】The results of this study demonstrate that DNA methylation plays a role in the wing development of B. mori by regulating autophagy. Our results provide the experimental evidence for the regulation of DNA methylation on the insect development.