Type

Journal Article

Authors

Martin Clynes
Niall Barron
Paula Meleady
Michael Henry
Nga T Lao
Orla Coleman
Alan Costello

Subjects

Biology

Topics
cho cells micrornas chinese hamster ovary cells proteomics cell density cricetulus animals ubiquitin thiolesterase

Depletion of endogenous miRNA-378-3p increases peak cell density of CHO DP12 cells and is correlated with elevated levels of ubiquitin carboxyl-terminal hydrolase 14. (2018)

Abstract miRNAs are potent molecular regulators of cellular behaviour. The manipulation of these small non-coding RNAs has been used to enhance industrially relevant phenotypes in Chinese Hamster Ovary (CHO) cells. We investigated the stable depletion of six miRNAs; miR-204-5p, 338-3p, 378-3p, 409-3p, 455-3p and 505-3p, robustly associated with cell growth rate from a previous profiling study. Inhibition of endogenous miR-378-3p function by miRNA-sponge-decoy improved peak cell density by 59%. Quantitative label free LC-MS/MS proteomic analysis of the fractionated cell cultures at day 4 and 8 of batch culture found 216 cytosolic and 114 membrane-associated proteins differentially expressed with stable miR-378-3p depletion. qRT-PCR of 8 genes; Clic4, Hnrnpa1, Prdx1, Actn4, Usp14, Srxn1, Canx and Gnb1, with unidirectional differential protein expression over the two time points of analysis was carried out. In-silico predictive algorithms; TargetScan and miRDB, were used to decipher possible direct targets of miR-378-3p. The Ubiquitin carboxyl-terminal hydrolase 14 (Usp14) protein was identified in the cytosolic fractions at both timepoints as differentially expressed with an increased abundance of 1.58-fold in the miR-378-3p depleted cells on day 8. Usp14 is a deubiquitinase (DUB) with previous reports of its up-regulation leading to increased proliferation of cancer cells. Overexpression of Usp14 in CHO cells had significant effects on cell growth supporting a role for Usp14 in the increased peak cell density seen with miR-378-3p depletion. This study highlights miR-378-3p as a novel engineering candidate for improving CHO cell growth. The use of subcellular fractionation also improved proteome coverage in the identification of novel miRNA targets.
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Full list of authors on original publication

Martin Clynes, Niall Barron, Paula Meleady, Michael Henry, Nga T Lao, Orla Coleman, Alan Costello

Experts in our system

1
Martin Clynes
Dublin City University
Total Publications: 232
 
2
Niall Barron
Dublin City University
Total Publications: 60
 
3
Paula Meleady
Dublin City University
Total Publications: 115
 
4
Michael Henry
Dublin City University
Total Publications: 99
 
5
Nga T Lao
Dublin City University
Total Publications: 15
 
6
Orla Coleman
Dublin City University
Total Publications: 12
 
7
Alan Costello
Dublin City University
Total Publications: 11