Biotechnology Core Laboratory NIDDK, NIH, Building 14A, Bethesda, MD 20892, USA.
Metab Eng. 2013 Mar;16:87-94. doi: 10.1016/j.ymben.2012.12.004. Epub 2013 Jan 29.
MiRNAs have been shown to be involved in regulation of multiple cellular processes including apoptosis. Since a single miRNA can affect the expression of several genes, the utilization of miRNAs for apoptosis engineering in mammalian cells can be more efficient than the conventional approach of manipulating a single gene. Mmu-miR-466h-5p was previously shown to have a pro-apoptotic role in CHO cells by reducing the expression of several anti-apoptotic genes and its transient inhibition delayed both the activation of Caspase-3/7 and the loss of cell viability. The present study evaluates the effect of stable inhibition of mmu-miR-466h-5p in CHO cells on their ability to resist apoptosis onset and their production properties. The expression of mmu-miR-466h-5p in the engineered anti-miR-466h CHO cell line was significantly lower than in the negative control and the parental CHO cells. These engineered cells reached higher maximum viable cell density and extended viability compared with negative control and parental CHO cells in batch cell cultures which resulted in the 53.8% and 41.6% increase of integral viable cells. The extended viability of anti-miR-466h CHO cells was the result of delayed Caspase-3/7 activation by more than 35h, and the increased levels of its anti-apoptotic gene targets (smo, stat5a, dad1, birc6, and bcl2l2) to between 2.1- and 12.5-fold compared with the negative control CHO in apoptotic conditions. The expression of secreted alkaline phosphatase (SEAP) increased 43% and the cell-specific productivity increased 11% in the stable pools of anti-miR-466h CHO compared with the stable pools of negative control CHO cells. The above results demonstrate the potential of this novel approach to create more productive cell lines through stable manipulation of specific miRNA expression.
miRNAs 被证明参与多种细胞过程的调节，包括细胞凋亡。由于单个 miRNA 可以影响几个基因的表达，因此在哺乳动物细胞中利用 miRNA 进行细胞凋亡工程可以比传统的操纵单个基因的方法更有效。Mmu-miR-466h-5p 先前被证明通过降低几种抗凋亡基因的表达在 CHO 细胞中具有促凋亡作用，其瞬时抑制延迟了 Caspase-3/7 的激活和细胞活力的丧失。本研究评估了稳定抑制 mmu-miR-466h-5p 在 CHO 细胞中对其抵抗凋亡起始能力及其生产特性的影响。工程化抗 miR-466h CHO 细胞系中 mmu-miR-466h-5p 的表达明显低于阴性对照和亲本 CHO 细胞。与阴性对照和亲本 CHO 细胞相比，这些工程细胞在批式细胞培养中达到了更高的最大活细胞密度和延长的存活时间，导致整体活细胞增加了 53.8%和 41.6%。抗 miR-466h CHO 细胞的延长存活时间是由于 Caspase-3/7 的激活延迟了超过 35 小时，并且其抗凋亡基因靶标（smo、stat5a、dad1、birc6 和 bcl2l2）的水平增加了 2.1-至 12.5 倍与凋亡条件下的阴性对照 CHO 相比。与阴性对照 CHO 细胞的稳定池相比，稳定池中的抗 miR-466h CHO 细胞的分泌碱性磷酸酶（SEAP）表达增加了 43%，细胞特异性产率增加了 11%。这些结果表明，通过稳定操纵特定 miRNA 的表达，这种新方法有可能创造出更具生产性的细胞系。
