Abstract
MicroRNAs are short non-coding RNAs that play an important role in the regulation of gene expression. Hence, microRNAs are considered as potential targets for engineering of Chinese hamster ovary (CHO) cells to improve recombinant protein production. Here, we analyzed and compared the microRNA expression patterns of high, low, and non-producing recombinant CHO cell lines expressing two structurally different model proteins in order to identify microRNAs that are involved in heterologous protein synthesis and secretion and thus might be promising targets for cell engineering to increase productivity. To generate reproducible and comparable data, the cells were cultivated in a bioreactor under steady-state conditions. Global microRNA expression analysis showed that mature microRNAs were predominantly upregulated in the producing cell lines compared to the non-producer. Several microRNAs were significantly differentially expressed between high and low producers, but none of them commonly for both model proteins. The identification of target messenger RNAs (mRNAs) is essential to understand the biological function of microRNAs. Therefore, we negatively correlated microRNA and global mRNA expression data and combined them with computationally predicted and experimentally validated targets. However, statistical analysis of the identified microRNA-mRNA interactions indicated a considerable false positive rate. Our results and the comparison to published data suggest that the reaction of CHO cells to the heterologous protein expression is strongly product- and/or clone-specific. In addition, this study highlights the urgent need for reliable CHO-specific microRNA target prediction tools and experimentally validated target databases in order to facilitate functional analysis of high-throughput microRNA expression data in CHO cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-014-5911-4) contains supplementary material, which is available to authorized users.
Highlights
Chinese hamster ovary (CHO) cells are the most frequently applied expression system for the production of therapeutic proteins, mainly because of their ability to grow in suspension and to secrete complex recombinant proteins that are correctly processed
This was mainly achieved by vector, media, and process optimizations, and, cell engineering has been applied to improve the productivity of recombinant CHO cells
The CHO cell lines were cultivated in a continuous process to establish steady-state conditions
Summary
Chinese hamster ovary (CHO) cells are the most frequently applied expression system for the production of therapeutic proteins, mainly because of their ability to grow in suspension and to secrete complex recombinant proteins that are correctly processed. A multitude of strategies to engineer apoptosis resistance, cell proliferation, product secretion, or cell metabolism have been described and are comprehensively reviewed elsewhere (Kim et al 2012) These approaches often include the stable overexpression of one or more genes, which constitutes an additional burden to the translational machinery of the cell. A single miRNA can repress hundred different mRNAs and thereby regulates entire gene networks (Hobert 2008) They play crucial roles in a wide range of biological processes including development, proliferation, differentiation, apoptosis, and metabolism (Bartel 2004; He and Hannon 2004). In CHO cells, it has already been shown that miRNAs can be utilized to improve growth (Jadhav et al 2012), apoptosis resistance (Druz et al 2013), and specific productivity (Barron et al 2011; Jadhav et al 2014; Strotbek et al 2013)
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