CHO细胞中DNMT3A对ATF3基因甲基化的影响

Effect of DNMT3A on the Methylation of ATF3 Gene in CHO Cells

转录激活因子3(ATF3)是一种早期应激反应基因,能够影响细胞增殖、分化、转化和凋亡。中国仓鼠卵巢细胞(CHO)是重组蛋白、单克隆抗体等生物大分子药物生产的主要宿主细胞之一。ATF3能够促进肿瘤细胞的增殖及抗凋亡等作用,理论上可对CHO细胞发挥调节作用。因此本试验对CHO细胞中ATF3的甲基化水平进行深入研究,通过重亚硫酸盐测序法检测CHO-K1细胞中ATF3的甲基化水平,发现ATF3启动子区域的胞嘧啶-磷酸-鸟嘌呤(CpG)甲基化高达90%。为了进一步研究ATF3甲基化的上游调控机制,构建了敲除DNA甲基转移酶3A(DNMT3A)的CHO-K1来研究DNMT3A对ATF3甲基化水平的影响。结果表明,敲除DNMT3A后,CHO-K1细胞中的ATF3甲基化水平降低至0,这充分证明了DNMT3A对ATF3的甲基化的调控作用,为CHO细胞的ATF3基因调控研究以及后续CHO细胞株改造提供了理论依据。

Activate transcription factor 3(ATF3) is an early stress response gene that can affect the proliferation,differentiation,transformation and apoptosis of cells.Chinese hamster ovary(CHO) cell is one of the main host cells for the production of recombinant protein,monoclonal antibody and other biological macromolecular drugs.ATF3 can promote tumor cell proliferation and anti-apoptosis,and ATF3 may have a promoting effect on the growth of CHO cells.Therefore,we conducted an in-depth study on the methylation level of ATF3 in CHO cells.The methylation level of ATF3 in CHO-K1 cells was detected by bisulfite sequencing,and it was found that the cytosine-phosphoric acid-guanine(CpG) methylation in the promoter region of ATF3 was as high as 90%.In order to study the upstream regulatory mechanism of ATF3 methylation,CHO-K1 knocked out DNA methyl transferase 3A(DNMT3A) was constructed to study the effect of DNMT3A on the level of ATF3 methylation.The results showed that after knocking out DNMT3A,the ATF3 methylation level in CHO-K1 cells was reduced to 0,which proved the regulatory effect of DNMT3A on ATF3 methylation.This paper provides a theoretical basis for the study of ATF3 gene regulation in CHO cells and the subsequent transformation of CHO cell lines.