MicroRNAs for Enhancement of CHO Cell Proliferation and Stability: Insights from Neuroblastoma Studies - MicroRNAs as Tools in Biopharmaceutical Production - page 91

mapping to primary RA response genes. RA causes a co-repressor complex, which
includes the nuclear receptor co-repressor 2 ( NCOR2 ) protein, to be displaced from
the RAR heterodimer complex by a co-activator complex. It is now clear that a num-
ber of miRNAs are significantly up-regulated in response to retinoic acid treatment
of neuroblastoma cell lines, indicating a potential role in the process of differen-
tiation (Beveridge et al. 2009 ; Chen et al. 2010 ; Chen and Stallings 2007 ; Foley
et al. 2011 ;Leetal. 2009 ; Meseguer et al. 2011 ; Ragusa et al. 2010 ). In particular,
functional studies involving ectopic over-expression of miR-125b, miR-10a/b, and
miR-214 indicate that these miRNAs promote neurite out-growth and the activation
of markers of differentiation such as GAP43 and TUBB (Chen et al. 2010 ; Foley et al.
2011 ;Leetal. 2009 ). Conversely, inhibition of endogenous miR-7 and miR-18a pro-
motes neuroblastoma cell differentiation in vitro . Remarkably, miR-10a/b targeting
of the NCOR2 mRNA, with a corresponding decrease in NCOR2 protein, is solely
responsible for triggering the commencement of differentiation. It was demonstrated
by Foley et al. ( 2011 ) that the NCOR2 co-repressor complex, in addition to being
displaced by RA, must also decrease via a miRNA-mediated mechanism. The dis-
placement and subsequent decrease in NCOR2 leads to the activation of primary
retinoic acid responsive genes, which in turn launch a cascade of indirect effects
that lead to a differentiated phenotype. The role of miRNAs in neuroblastoma cell
differentiation is clearly very complex and a great amount of further investigation is
required to elucidate these interesting and intricate regulatory networks.

6.5 miRNAs Involved with DNA Methylation

The hypermethylation of gene promoter regions plays a major role in inactivating
tumor suppressor genes in the development of cancer (Kulis and Esteller 2010 ).
Recently, it was also demonstrated that genome-wide DNA methylation alterations
occur during the process of retinoic acid induced neuroblastoma differentiation,
which appear to be mediated by miRNAs that target DNA methyltransferase (Das
et al. 2010 ). In studies carried out by Das et al., a large set of genes were deter-
mined to be de-methylated in response to RA, consistent with the decrease in DNA
methyltransferase activity. To determine a potential mechanism for the decrease
in DNMT expression, a miRNA expression screen was performed pre- and post-
RA which identified 17 up-regulated miRNAs. One of the up-regulated miRNAs,
miR-152, was determined to directly target DNMT1 , which maintains methylation
patterns during cell division with a preference for hemi-methylated DNA (Hermann
et al. 2004 ). Interestingly, other miRNAs up-regulated in response to RA, such as
miR-125a/b and miR-26a/b, are predicted to target DNMT3B , and thus might also
contribute to changes in the DNA methylome during differentiation.
An example of one of the genes that was demethylated and over expressed in sev-
eral neuroblastoma cell lines treated with ATRA was nitric acid synthetase ( NOS1)
(Das et al. 2010 ), which catalyzes the generation of nitric oxide and has been im-
plicated in neuroblastoma cell proliferation and differentiation (Ciani et al. 2004 ).