MicroRNAs for Enhancement of CHO Cell Proliferation and Stability: Insights from Neuroblastoma Studies - MicroRNAs as Tools in Biopharmaceutical Production - page 90

artificially increasing miR-17-5p-92 levels in CHO cells might further improve the
characteristics of the line for bioprocessing purposes.

6.3 Apoptosis Promoting miRNAs

In general, more miRNAs appear to be down-regulated than up-regulated in can-
cers, indicating that they have major tumor suppressive effects (Bray et al. 2009 ).
A number of miRNAs cause a variety of cancer cell types to undergo apoptosis
when ectopically over-expressed, including both miR-34a and miR-184 in neurob-
lastoma. My research group was the first to demonstrate that miR-34a has tumor
suppressor functions in cancer (Welch et al. 2007 ). MiR-34a maps to a region on
human chromosome 1p which is frequently deleted in neuroblastoma tumors, re-
sulting in lower expression of this miRNA. Ectopic over-expression of this miRNA
in neuroblastoma cell lines leads to the arrest of cell proliferation and the induction
of a caspase-mediated apoptotic pathway. miR-34a targets several genes with pro-
proliferative or anti-apoptotic effects, including the E2F3 and MYCN transcription
factors (Cole et al. 2008 ; Wei et al. 2008 ; Welch et al. 2007 ), BCL2 (Bommer et al.
2007 ), CCND1 (Sun et al. 2008 ) and CDK6 (Sun et al. 2008 ). Intriguingly, miR-34
family members are directly up-regulated by the tumor suppressive p53 transcription
factor, which is mutated in greater than 50 % of all cancers (Bommer et al. 2007 ;
Chang et al. 2007 ;Heetal. 2007a ; Raver-Shapira et al. 2007 ; Tarasov et al. 2007 ).
MiR-184 is another miRNA with pro-apoptotic effects in neuroblastoma, as orig-
inally demonstrated by Chen et al (Chen and Stallings 2007 ). Remarkably, even
though miR-184 is predicted to target 100s of mRNA sequences, Foley et al. ( 2010 )
determined that the molecular mechanism leading to apoptosis was due solely to the
targeting of the pro-survival serine-threonine kinase AKT2 . siRNA-mediated knock-
down of AKT2 by itself resulted in apoptosis, and an apoptotic phenotype caused by
miR-184 targeting of AKT2 could be rescued by co-transfection of miR-184 with an
AKT2 expression plasmid lacking a 3 UTR target site.
MiR-34a has many conserved binding sites between vertebrate genomes, indicat-
ing that the engineering of CHO cells with reduced levels of miR-34a could be of
interest. The miR-184/ AKT2 interaction site, on the other hand, is poorly conserved,
and might not be of interest for further studies in CHO.

6.4 miRNAs Regulating Cell Differentiation

An important feature of neuroblastoma cells, and other cancer cell types, is their
ability to undergo differentiation following treatment with retinoic acid or other dif-
ferentiating agents (Stallings et al. 2011 ). Retinoic acid (RA) is a vitamin A derivative
which activates gene transcription by binding to retinoic acid receptors (RAR) which
are attached to retinoic acid response elements, which are short DNA sequence motifs