Kelly Paul S, Breen Laura, Gallagher Clair, Kelly Shane, Henry Michael, Lao Nga T, Meleady Paula, O'Gorman Donal, Clynes Martin, Barron Niall
National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland.
Centre for Preventative Medicine, School of Health and Human Performance, Dublin City University, Dublin, Ireland.
Biotechnol J. 2015 Jul;10(7):1029-40. doi: 10.1002/biot.201500101. Epub 2015 Jun 24.
microRNA engineering of CHO cells has already proved successful in enhancing various industrially relevant phenotypes and producing various recombinant products. A single miRNA's ability to interact with multiple mRNA targets allows their regulatory capacity to extend to processes such as cellular metabolism. Various metabolic states have previously been associated with particular CHO cell phenotypes such as glycolytic or oxidative metabolism accommodating growth and productivity, respectively. miR-23 has previously been demonstrated to play a role in glutamate metabolism resulting in enhanced oxidative phosphorylation through the TCA cycle. Re-programming cellular bioenergetics through miR-23 could tip the balance, forcing mammalian production cells to be more productive by favoring metabolic channelling into oxidative metabolism. CHO clones depleted of miR-23 using a miR-sponge decoy demonstrated an average ∼three-fold enhanced specific productivity with no impact on cell growth. Using a cell respirometer, mitochondrial activity was found to be enhanced by ∼30% at Complex I and II of the electron transport system. Additionally, label-free proteomic analysis uncovered various potential novel targets of miR-23 including LE1 and IDH1, both implicated in oxidative metabolism and mitochondrial activity. These results demonstrate miRNA-based engineering as a route to re-programming cellular metabolism resulting in increased productivity, without affecting growth.
对中国仓鼠卵巢（CHO）细胞进行微小RNA工程改造已被证明在增强各种与工业相关的表型以及生产各种重组产品方面是成功的。单个微小RNA与多个信使核糖核酸（mRNA）靶标的相互作用能力使其调控能力能够扩展到细胞代谢等过程。以前，各种代谢状态分别与特定的CHO细胞表型相关，例如糖酵解或氧化代谢分别适应生长和生产力。以前已经证明，微小RNA-23（miR-23）在谷氨酸代谢中起作用，通过三羧酸循环（TCA循环）增强氧化磷酸化。通过miR-23对细胞生物能量学进行重新编程可能会打破平衡，通过促进代谢导向氧化代谢，迫使哺乳动物生产细胞提高生产力。使用微小RNA海绵诱饵耗尽miR-23的CHO克隆显示，比生产率平均提高了约三倍，而对细胞生长没有影响。使用细胞呼吸计发现，电子传输系统复合体I和II处的线粒体活性提高了约30%。此外，无标记蛋白质组学分析发现了miR-23的各种潜在新靶标，包括LE1和异柠檬酸脱氢酶1（IDH1），两者都与氧化代谢和线粒体活性有关。这些结果表明，基于微小RNA的工程改造是一种重新编程细胞代谢从而提高生产力而不影响生长的途径。
