A General Introduction to MicroRNAs,Their Investigation and Exploitation in CHO Cell Lines - MicroRNAs as Tools in Biopharmaceutical Production - page 9

in IgG producing and parental cell lines and found that miR-221 and miR-222 were
significantly down-regulated in the antibody producing DG44 cell lines. However,
there was no specific correlation between the volumetric IgG productivity and the
relative expression of miR-221 and miR-222.
A further difficulty in CHO based microRNA studies remains that several of the
target prediction software such as TargetScan still restrict their search to the human
and mouse 3 UTRs and systematic investigation of targets for CHO miRNA is often
hampered by the lack of an experimental database for the Chinese hamster. However,
other software such as TargetRank allow the user to enter unknown sequences. If the
degree of conservation of a particular miRNA between hamster and other vertebrates
is high, one would predict that there is some degree of overlap in their target speci-
ficity. However, because of the different parameters and statistical techniques used
in the current target prediction software it is sometimes difficult to define the ranking
and scoring of targets. As an example, if one looks in either the mouse or the human
genome for the putative targets of let-7f: TargetScan (http://www.targetscan.org/) and
TargetRank (http://genes.mit.edu/targetrank/) agree on the target genes ranked 1 and
3 but disagree on the target gene ranked 2. However, the outcome is similar whether
the search is undertaken with the human or the mouse microRNA. On the other hand,
the top three predicted targets of miR-17, a broadly conserved microRNA, do not
match when the query is run using the same research tools. As discussed above,
because of the disparate nature of the target prediction it is essential to validate the
predicted miRNA:mRNA physical interaction experimentally.
Investigators of CHO microRNA biology can use a number of approaches to
validate the influence of perturbation on cell phenotype as discussed above. If a given
mRNA is a true target of a specific miRNA, then perturbation of the mRNA:miRNA
ratio should lead to a change in the amount of protein encoded by the target mRNA.
Therefore, routine techniques such as Western analysis can be used to validate the
functional importance of a given miRNA/mRNA target pair. Alternatively, ELISA
experiments could also be utilized to quantify differences in protein expression. The
target gene levels can also be verified by real-time RT-PCR. If the mRNA level
is reduced, this suggests that the mechanism of protein downregulation involves
mRNA degradation by deadenylation and exonucleolytic attack. However, if the
miRNA does not primarily regulate gene expression by degradation of its target it
may be more difficult to detect changes in mRNA levels but protein levels should
still be altered due to translation attenuation.
To assess the impact of miR manipulation on protein expression, a few miRNA
target validation reporter gene systems based on luminescence (Firefly (Tanguay and
Gallie 1996 ), Renilla (Kong et al. 2008 ) or Gaussia luciferase (Kim et al. 2008 )) have
been developed for the quantitative assessment of the degree of post-transcriptional
repression. Commonly, the 3 UTR of the predicted target is cloned downstream of
the luciferase marker and the construct is transfected in the host cells with varying
amounts of microRNAs (which can be upregulated from plasmid DNA or reduced
using sponge vectors). Finally, the quantity of light emitted upon substrate cleavage
is measured for each sample.