MicroRNAs for Enhancement of CHO Cell Proliferation and Stability: Insights from Neuroblastoma Studies - MicroRNAs as Tools in Biopharmaceutical Production - page 93

et al. 2007 ). siRNA-mediated inhibition of one T-UCR in a colon cancer cell line
was determined to have anti-proliferative effects (Calin et al. 2007 ). More recently,
Scaruffi et al. ( 2009 ) and Mestdagh et al. ( 2010b ) have reported that T-UCRs are dif-
ferentially expressed in clinically favorable versus unfavorable neuroblastoma tumor
subtypes, indicating a potential role for these non-coding sequences in the develop-
ment of this disease. Interestingly, there is also preliminary evidence indicating that
T-UCRs are regulated by miRNAs (Scaruffi et al. 2009 ; Calin et al. 2007 ), adding an-
other layer of complexity to an already complex miRNA regulatory network. In some
instances, larger non-coding RNAs could be acting as “sponges”, i.e. endogenous
competitors for miRNAs (Ebert and Sharp 2010 ).
Given that T-UCRs have very significant conservation between human and rodent
genomes, by definition, and that some T-UCRs have anti-proliferative effects on
human cancer cells, studies on these sequences in CHO cells would be of potential
interest for further improving the phenotypic characteristics of this cell line for protein
production.

6.8 The Mechanism of MicroRNA Action is not Completely

Understood

Although it is now definitively established that miRNAs target the 3 UTRs of large
numbers of mRNA sequences, resulting in either degradation of the mRNA or trans-
lational inhibition, there are a number of reports that indicate miRNAs also have
other mechanisms of action. For example, let-7 family members can apparently en-
hance the translational efficiency of target mRNA sequences in cells undergoing cell
cycle arrest, while inhibiting translation in actively proliferating cells (Vasudevan
et al. 2007 ). Let-7 is also an interesting family of miRNAs from the standpoint
of processing of precursor sequences into biologically active mature miRNAs. The
RNA binding proteins LIN28 and LIN28B can bind to Let-7 RNA precursors, block-
ing their bioprocessing by either Drosha or Dicer into mature sequences (Heo et al.
2009 ). Additionally, RNA binding proteins can bind to 3 UTR sequences and pro-
tect mRNA sequences from miRNA targeting (Kedde et al. 2007 ). The let-7 family
of miRNAs is highly conserved in diverse organisms, indicating that studies of this
family in CHO cells is warranted, particularly in view of the fact that these miRNAs
play major roles in cancer.
It is important to realize that miRNAs can interact with complementary sequences
in the 5 UTR and in the coding region of genes, in addition to 3 UTR regions
(Duursma et al. 2008 ; Orom et al. 2008 ), although a proteome-wide study by Baek
et al indicated that miRNA interaction with 3 UTRs had greater efficacy than the
coding regions (Baek et al. 2008 ). This is likely explained by experiments performed
by Gu et al. ( 2009 ), where the authors demonstrated that extending a coding region
through the 3 UTR of the miRNA target site by modification of the stop codon
resulted in the loss of a miRNAs ability to translationally inhibit the mRNA sequence.
This provides a biological reason for why most miRNA target sites are localized in