Biomedical Engineering Reference
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mRNA transcription, processes known to be reduced upon temperature reduction
(Roobol et al. 2009 ). Among the down-regulated candidate miRNAs, miR-7 stood
out with greater than 8-fold repression after temperature reduction. Due to its known
interactions with mRNAs of Pak1, which is a member of p21 activating kinases,
and insulin-growth factor receptor 1 (Jiang et al. 2010 ; Reddy et al. 2008 ), miR-7
was chosen for functional characterization by transient overexpression and knock-
down. Results from these experiments attributed miR-7 a role as active inhibitor of
cell growth upon overexpression, accompanied by an increase in recombinant pro-
tein production rates, thus mimicking the effects of biphasic cultivation strategies.
Down-regulation of miR-7 using antisense oligonucleotides, however, did not result
in increased growth of CHO cells suggesting redundant mechanisms in the gene
expression control of its targets.
Another recent study focused in detail on the dynamics of miRNA levels during
batch cultivation of CHO-K1 cells (Bort et al. 2011 ). Overall 118 miRNAs were iden-
tified that were significantly regulated during the course of a batch culture compared
to lag phase by exhibiting a > 1.5 fold change in expression level over time. Cluster
analysis of the expression profiles of these 118 candidates identified two principal
groups: (i) 43 miRNAs (36 %) with constant increase in transcript levels towards
stationary and decline phase, and (ii) 75 miRNAs (64 %) with peak levels during
exponential phase and subsequent down-regulation during stationary and decline
phase, therefore correlating with specific growth rate of cells during batch cultiva-
tion. Among the miRNAs sharing this profile, five distinct miRNA families (miRNAs
with high sequence similarity) and six miRNA clusters (miRNAs organized in close
genomic proximity and therefore belonging to the same transcriptional unit) were
identified that were represented by at least two or more of its members.
The expression pattern of miRNAs of the miR-17-92 cluster was of particular
interest, as this cluster has been previously implicated in the regulation of cell cy-
cle progression (Cloonan et al. 2008 ; Pickering et al. 2009 ), proliferation (Manni
et al. 2009 ) and oncogenicity (Olive et al. 2009 ). Several studies focusing on target
identification ofmiR-17-92 indicate that this cluster mediates its effect by negative
regulation of important cell cycle protein mRNAs such as CDKN1 A (p21), CDKN1
C (p57), or retinoblastoma 1 (Rb1), as well as apoptosis related protein mRNAs such
as phosphatase tensin homolog (PTEN), or BCL2-like-11 (reviewd by Grillari et al.
2010 ). Based on RNA-seq data generated from different CHO cell lines (Becker et al.
2011 ), the majority of the reported target sites for miR-17-92 could be identified in
CHO (Hackl et al. 2011 ), suggesting a similar function for miR-17-92 in CHO cells.
Therefore, elevated levels of miR-17-92—as observed during exponential growth of
CHO cells—could indirectly induce (or support) a proliferative state by suppressing
cell cycle inhibitors p21 and p57, thus leading to an activation of E2 F transcription
factor.
While this cluster of miRNAs was identified as a potential effector of CHO cell
growth and survival by separate analysis of miRNA array-data, an integrated analysis
of miRNA and mRNA microarray array data from CHO batch cultivation was also
reported (Bort et al. 2011 ). The results indicate that in particular the up-regulation of a
set of mRNAs during the late phases of batch cultivation where cell growth is slowing
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