The CHO miRNA Transcriptome - MicroRNAs as Tools in Biopharmaceutical Production - page 59

down to finally become arrested, may be enhanced or controlled by simultaneous
down-regulation of a group of miRNAs, which preferentially bind to these mRNAs.
This observed negative correlation in the expression of miRNAs and their predicted
mRNA targets point towards functional miRNA

∼ mRNA networks that regulate the

growth of CHO cells.

4.4.3 ApoptomiRs—miRNAs in Control of Survival and Death

of CHO Cells

Quite fittingly, the term apoptomiR was recently established to describe miRNAs that
can influence death and survival of a cell by regulating cellular apoptosis (Vecchione
and Croce 2010 ). This evolutionary conserved process, which enables an organism
to remove cells no longer useful for its function or even dangerous for its survival was
also shown to trigger the death of CHO cells in cultivation, for example in response
to nutrient depletion or accumulation of toxic metabolites (Arden and Betenbaugh
2004 ). Hence, several cell engineering approaches employed over-expression of
anti-apoptotic genes such as members of the BCL-2 superfamily of proteins, to
protect CHO cells from undergoing apoptosis and consequently allow for higher cell
densities and therefore space-time yields (Majors et al. 2008 , 2009 ). Since it is likely
that a combination of down-regulation and over-expression of specific pro- and anti-
apoptotic genes is the most promising approach to control apoptosis in CHO cells,
miRNAs, with their range of distinct targets and ability to stabilize cellular states,
might prove to be valuable tools in this endeavor.
So far, only one study has specifically addressed the relevance of miRNAs for
apoptosis in CHO cells by analyzing the changes in miRNA expression upon nutri-
ent depletion, which was shown to increase Caspase-3/7 activity and consequently
trigger apoptosis (Druz et al. 2011 ). Using microarray technology 70 miRNAs were
identified as either significantly up or down-regulated in cells exposed to nutrient
depleted media compared to fresh media controls. Among these, all 18 members of
the miR-297-669 cluster that were detected on the array were found to be strongly
induced in apoptotic versus control cells, with miR-466h being 452-fold induced
in cells exposed to nutrient depleted media. Such a high level of induction, which
is rarely observed for miRNAs, is possibly the result of miR-466h transcription or
processing being shut-down under normal conditions, thus suggesting an important
but also tightly controlled function for this miRNA: by in silico analyses of potential
miR-466h targets, nine genes with anti-apoptotic function such as BCL2 or BCL2L2
were identified, thus pointing towards a pro-apoptotic function of miR-466h (Druz
et al. 2011 ).
Druz et al. ( 2011 ) further went into functional characterization of miR-466h by
again inducing apoptosis in CHO cells via nutrient depletion, but at the same time
reducing miR-466h levels using antisense-inhibitor sequences. The suppression of
miR-466h induction upon nutrient depletion improved the viability of CHO cells