A functional high‐content miRNA screen identifies miR‐30 family to boost recombinant protein production in CHO cells

Fischer, Simon; Buck, Theresa; Wagner, Andreas; Ehrhart, C; Giancaterino, Julia; Mang, Samuel; Schad, Matthias; Mathias, Sven; Aschrafi, Armaz; Handrick, René; Otte, Kerstin

doi:10.1002/biot.201400306

Cited by 61 publications

(92 citation statements)

References 59 publications

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“…One plausible route might be post-transcriptional regulation by miRNAs, which is supported by the observation of numerous changes in miRNA expression in this study. Interestingly, miR-30 was found to be up-regulated in subclones characterized by high qP, which is in line with results from a recent high-throughput screen of miRNA overexpression in a CHO-SEAP cell line [65]. Therefore, a thorough integrative computational analysis of miRNA and mRNA expression data in the future could allow to identify miRNA~mRNA networks that support a high productivity state.…”

Section: Discussionsupporting

confidence: 82%

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

Harreither

Hackl

Pichler

et al. 2015

Biotechnology Journal

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Over the last three decades, product yields from CHO cells have increased dramatically, yet specific productivity (qP) remains a limiting factor. In a previous study, using repeated cell-sorting, we have established different host cell subclones that show superior transient qP over their respective parental cell lines (CHO-K1, CHO-S). The transcriptome of the resulting six cell lines in different biological states (untransfected, mock transfected, plasmid transfected) was first explored by hierarchical clustering and indicated that gene activity associated with increased qP did not stem from a certain cellular state but seemed to be inherent for a high qP host line. We then performed a novel gene regression analysis identifying drivers for an increase in qP. Genes significantly implicated were first systematically tested for enrichment of GO terms using a Bayesian approach incorporating the hierarchical structure of the GO term tree. Results indicated that specific cellular components such as nucleus, ER, and Golgi are relevant for cellular productivity. This was complemented by targeted GSA that tested functionally homogeneous, manually curated subsets of KEGG pathways known to be involved in transcription, translation, and protein processing. Significantly implicated pathways included mRNA surveillance, proteasome, protein processing in the ER and SNARE interactions in vesicular transport.

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Section: Discussionsupporting

confidence: 82%

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

Harreither

Hackl

Pichler

et al. 2015

Biotechnology Journal

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“…We recently conducted an unbiased functional miRNA screen in a recombinant CHO cell line assessing the impact of 1139 miRNAs on crucial cellular functions. 11 In this report, we demonstrate that each of these vital cellular pathways could be controlled by an unexpectedly large number of miRNAs, which may indicate a redundancy in pathway regulation guaranteeing survival of the cell. Since the pathways are to some degree interrelated, we found that subsets of miRNAs have both specific as well as overlapping functions.…”

Section: Introductionmentioning

confidence: 67%

“…Transfections of miRNA mimics and small interfering RNAs (siRNAs) were carried out essentially as described previously. 11 An entire murine miRNA mimics library (miRBase release 18) comprising 1139 different miRNAs was used for the transient miRNA screening (Qiagen, Hilden, Germany) (see also Supplementary Table S1). An Alexa Fluor Ò 647-labeled non-targeting siRNA, a non-labeled, scrambled siRNA (NT siRNA), an anti-SEAP siRNA (siSEAP) (all Qiagen) and a cell death control siRNA (siDeath) (kindly provided by Dr. Eric Lader, Qiagen) served as functional transfection controls.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, stable overexpression of pre-miR-30a in CHO cells was shown to enhance cell proliferation. 11 Conversely, oncogenes such as c-Myc, KRAS or growth factor receptors, whose overexpression is directly linked to uncontrolled cell growth and tumorigenesis, might have been targeted by miRNAs leading to diminished cell growth. In papillary thyroid cancer cells, let-7f has been detected to be down-regulated, whereas overexpression of let-7 was shown to inhibit cell growth, by targeting c-Myc.…”

Section: Control Of Cell Proliferation By Mirnasmentioning

confidence: 99%

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Unveiling the principle of microRNA-mediated redundancy in cellular pathway regulation

et al. 2015

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per minute; SEAP; secreted alkaline phosphatase; siRNA; small-interfering RNA Understanding the multifaceted nature of microRNA (miRNA) function in mammalian cells is still a challenge. Commonly accepted principles of cooperativity and multiplicity of miRNA function imply that individual mRNAs can be targeted by several miRNAs whereas a single miRNA may concomitantly regulate a subset of different genes. However, there is a paucity of information whether multiple miRNAs regulate critical cellular events and thereby acting redundantly. To gain insight into this notion, we conducted an unbiased high-content miRNA screen by individually introducing 1139 miRNA mimics into Chinese hamster ovary (CHO) cells. We discovered that 66% of all miRNAs significantly impacted on proliferation, protein expression, apoptosis and necrosis. In summary, we provide evidence for a substantial degree of redundancy among miRNAs to maintain cellular homeostasis.

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“…To identify miRNAs able to increase recombinant protein production in Chinese hamster ovary (CHO) cells, the prototype mammalian cell line for production of biopharmaceuticals, Fischer and collaborators 43 performed a miRNA screening using a library of 1139 human miRNA mimics. In this study, the miR-30 family was recognized as a potent enhancer of protein production in CHO cells.…”

Section: V12mentioning

confidence: 99%

MicroRNA Screening and the Quest for Biologically Relevant Targets

Eulálio

Mano

2015

SLAS Discovery

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MicroRNAs (miRNAs) are a class of genome-encoded small RNAs that post-transcriptionally regulate gene expression by repressing target transcripts containing partially or fully complementary binding sites.Despite their relatively low number, miRNAs have been shown to directly regulate a large fraction of the transcriptome. In agreement with their pervasive role in the regulation of eukaryotic gene expression, miRNAs have been implicated in virtually all biological processes, including different pathologies.The use of screening technologies to systematically analyze miRNA function in cell-based assays offers a unique opportunity to gain new insights into complex biological and disease-relevant processes. Given the low complexity of the miRNome and the similarities to small interfering RNA (siRNA) screening experimental approaches, phenotypic screening using genome-wide libraries of miRNA mimics or inhibitors is not, per se, technically challenging. The identification of miRNA targets and, more importantly, the characterization of their mechanisms of action through the identification of the key targets underlying observed phenotypes remain the major challenges of this approach.This article provides an overview of cell-based screenings for miRNA function that were performed in different biological contexts. The advantages and limitations of computational and experimental approaches commonly used to identify miRNA targets are also discussed.

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A functional high‐content miRNA screen identifies miR‐30 family to boost recombinant protein production in CHO cells

Cited by 61 publications

References 59 publications

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

Microarray profiling of preselected CHO host cell subclones identifies gene expression patterns associated with in‐creased production capacity

Unveiling the principle of microRNA-mediated redundancy in cellular pathway regulation

MicroRNA Screening and the Quest for Biologically Relevant Targets

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