MicroRNAs as Engineering Targets: Pathway Manipulation to Impact Bioprocess Phenotypes - MicroRNAs as Tools in Biopharmaceutical Production - page 75

of pyruvate to lactate, reduced glycolytic flux and the specific lactate production
rate in CHO cells (Kim and Lee 2007 ). However, despite the reduced lactate accu-
mulation and increased rate of oxidative phosphorylation, CHO cells became more
susceptible to oxidative stress after knock-down of the LDH-A (Jeong et al. 2004 ).
Korke et al. showed that a lowered ratio of glucose consumption to lactate production
in hybridoma cells culture was the result of global changes in gene expression at the
transcription level (Korke et al. 2004 ) and may be in part due to regulation of genes
by miRNAs (Muller et al. 2008 ).
Another tool to investigate metabolic phenotypes is a metabolomics approach cou-
pled with 13 C-flux analysis of cellular metabolism and its regulation in mammalian
cells. This approach offers the quantification of internal metabolic fluxes, providing
researchers with comprehensive information on cellular energetics (Zamboni and
Sauer 2009 ).
Several miRNAs have been related to metabolic control. (1) miR-375 was shown
to regulate glucose homeostasis and glucose-mediated insulin secretion in pancre-
atic endocrine cell lines by targeting the myotrophin (Mtpn) gene (Poy et al. 2007 ).
miR-375 was also shown to down-regulate 3 -phosphoinositide-dependent protein
kinase-1 (PDK-1), resulting in decrease of insulin gene expression in primary rat
islets (El Ouaamari et al. 2008 ). Over-expression of miR-375 decreased glucose-
induced insulin secretion with no effects on glucose-stimulated ATP production or
intracellular Ca 2 + levels (Gauthier and Wollheim 2006 ); (2) miR-124a, miR-107,
miR-30d were up-regulated, and miR-296, miR-484, miR-690 were down-regulated
at high glucose conditions in pancreatic beta cells. Over-expression of miR-30d in-
creased insulin gene expression indirectly, but had no effects on insulin secretion
(Tang et al. 2009 ); (3) miR-122 was shown to be involved in regulation of choles-
terol and lipid homeostasis in mice (Esau et al. 2006 ; Krutzfeldt and Stoffel 2006 );
(4) miR-29b involvement in amino acid metabolism was shown in HEK293 cells. This
miRNA controlled the branched amino acid (BCAA) metabolism by targeting the
branched-chain -ketoacid dehydrogenase (BCKD) enzyme, known to catalyze the

irreversible step in BCAA catabolism (Mersey et al. 2005 ); (5) Gao and colleagues
showed the effect of miR-23a/b in human B lymphoma cells on the regulation of
glutamine metabolism by targeting mitochondrial glutaminase (GLS) expression. It
was also shown that miR-23a/b is subject to c-MYC regulation (Gao et al. 2009 );
(6) miR-378 ∗ , was shown to induce the metabolic shift in breast cancer cells by target-
ing estrogen related receptor (ERR

α

(GABPA), two key
regulators of energy metabolism. Over-expression of miR-378 ∗ reduced the activity
of the TCA cycle, rendering the cells less dependent on oxidative phosphorylation
and causing increased lactate production (Eichner et al. 2010 ).
Some metabolism-related miRNAs discussed above (miR-23a/b, -29b, -30d,
-107, -122, -296, -484, -378 ∗ ) were already reported in CHO cells (Hackl et al.
2011 ; Johnson et al. 2011 ). miR-dependent regulation of metabolic pathways is rela-
tively new; therefore, comprehensive analysis of miR-mediated control of metabolic
enzymes and fluxes and their effects on metabolic phenotypes, coupled with the
investigation of CHO-specific miRs and gene targets, needs to be conducted.

γ ), and GA-binding protein α