MicroRNAs as Engineering Targets: Pathway Manipulation to Impact Bioprocess Phenotypes - MicroRNAs as Tools in Biopharmaceutical Production - page 71

Sauerwald et al. 2006 ; Tey et al. 2000 ). Also over-expression of viral homologs of
bcl-2 such as E1B-19K was shown to reduce apoptosis in mammalian cell cultures
(Figueroa et al. 2007 ; Mercille and Massie 1999 ; Sauerwald et al. 2002 ).
RNAi silencing techniques against pro-apoptotic genes have been successfully
applied. Stable inhibition of Bax and Bak with shRNA vectors, and silencing of the
apoptosis linked gene Alg-2 and the transcriptional factor Requiem in CHO cells de-
layed apoptosis, increased cell viability and improved interferon- production (Lim

et al. 2006 ; Wong et al. 2006 ). Also, the simultaneous down-regulation of caspase-3
and caspase-7 expression improved cell viability and thrombopoietin production in
CHO cells after sodium butyrate treatment (Sung et al. 2007 ).
miRNA involvement in apoptosis regulation was initially studied in peripheral
blood cells of people diagnosed with chronic lymphocytic leukemia (CLL) where
deletion of miR-15 and miR-16 genes was reported in the majority of patients (Calin
et al. 2002 ). Later studies revealed that these miRNAs were promoting apoptosis
in malignant B cells by targeting Bcl-2 expression at the post-transcriptional level
(Cimmino et al. 2005 ). miR-21 (in addition to its involvement in cell growth regula-
tion) was found to be up-regulated in several human cancers and was characterized
as an oncogenic miR. Silencing of miR-21 expression in glioblastoma cells led to
increased apoptosis by activation of caspase 3 and 7 (Chan et al. 2005 ; Meng et al.
2006 ;Sietal. 2007 ).
Cheng et al. have identified several miRNAs involved in apoptosis regulation by
using large-scale antisense miRs inhibition. The inhibition of miR-1d, 7, 148, 204,
210, 216 and 296 in HeLa cells increased apoptosis by activation of caspase 3, while
the inhibition of miR-214 had the opposite effect (Cheng et al. 2005 ).
Li et al. have investigated the pro-apoptotic role of miR-204 in human trabecular
meshwork (HTM) cells. This miR directly targeted several anti-apoptotic genes and
increased cell susceptibility to apoptosis (Li et al. 2011 ).
Other miRNAs have a role in the regulation of apoptosis: (1) miR-218 was found
to be involved in NF-kappaB response and apoptosis induction by targeting ex-
pression of the ECOP gene (Gao et al. 2010 ); (2) miR-1 and miR-133 produced
opposing effects on apoptosis induced by oxidative stress in rat cardiomyocytes
(Xu et al. 2007 ). miR-1 had a pro-apoptotic function in response to oxidative stress
by targeting heat shock proteins HSP60 and HSP70 and miR-133 seemed to have
an anti-apoptotic role by repressing caspase 9 gene expression; (3) miR-34 family
members were found to function as potent mediators of the p53-induced apoptotic
pathway by targeting anti-apoptotic genes including Bcl-2, and were also found to
participate in a positive feedback loop of p53 activation via increased acetylation
by targeting SIRT1 deacetylase (Hermeking 2010 ); (4) miR-30 was shown to affect
the levels of the Ubc9 and ITGB3 genes in breast tumor-initiating cells which re-
stricted their self-renewing capacity and targeted them for apoptosis (Yu et al. 2010 );
(5) miR-10a was shown to participate in the TRAIL-induced apoptosis pathway lead-
ing to caspase 3 activation in human lung carcinomas (Ovcharenko et al. 2007 ); (6)
the members of let-7 miRs family, let-7c and let-7g, were shown to target Bcl-x L
directly and Mcl-1 indirectly which led to caspase-3/7 activation and apoptosis
induction in hepatocytes (Shimizu et al. 2010 ).

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