miRNA-20a作为CHO-K1细胞抗凋亡调控靶标的研究

Study on miRNA-20a as targets for anti-apoptosis in CHO-K1 cells

通过分析miRNA表达谱发现,miRNA-20a在凋亡CHO-K1细胞中的表达降低。通过构建稳定表达miRNA-20a的重组细胞株,分析该重组细胞的抗凋亡能力。分别构建携带miRNA-20a前体序列或反义序列的重组慢病毒,转染CHO-K1细胞,得到miRNA-20a功能获得或功能抑制的重组细胞。无血清过度消耗培养液应激环境下培养重组细胞,分析靶基因E2F1的表达水平,通过细胞活力和Caspase3/7活性分析评价细胞的抗凋亡能力。结果显示,miRNA-20a过表达的细胞株,E2F1的表达受到显著抑制,在无血清过度消耗的应激环境下,细胞活力显著提高,Caspase3/7活性降低。相反,miRNA-20a抑制表达的细胞株中,E2F1表达增加,而细胞活性显著降低,抗凋亡能力显著下降。上述结果提示,miRNA-20a有望作为哺乳动物宿主细胞的遗传重构靶标,构建耐受应激环境的抗凋亡细胞。

The miRNA microarray analysis showed that the miR-20 aexpression was significantly downregulated in apoptotic CHO-K1 cells,which suggested that miRNA-20 awere potential target for genetic engineering cells to enhance apoptosis resistance.The genetic recombinant CHO-K1 cells with miRNA-20 astable expression or inhibiting expression were constructed using the recombinant lentivirus vectors with pre-miR-20 asequences or anti-miR-20 asequences.To evaluate anti-apoptosis ability of recombinant cells,the cells were cultured in spent medium without serum,then the expression level of target gene E2F1,cell viability and Caspase 3/7activity were analysed.The results show that the expression of E2F1 and Caspase 3/7activity are significantly inhibited,consequently the cells viability is improved.In contrast,the anti-apoptosis anbility is weakened in recombinant CHO-K1 cells with inhibited miR-20 aexpression.These results suggest that stable miR-20 aexpression is beneficial to maintain viability of cells exposed to full-stress culture and miR-20 amay become a target for developing new technologies in enhancing the resistance of mammalian cells to shear stress.