Stable inhibition of mmu-miR-466h-5p improves apoptosis resistance and protein production in CHO cells

Abstract

MiRNAs have been shown to be involved in regulation of multiple cellular processes including apoptosis. Since a single miRNA can affect the expression of several genes, the utilization of miRNAs for apoptosis engineering in mammalian cells can be more efficient than the conventional approach of manipulating a single gene. Mmu-miR-466h-5p was previously shown to have a pro-apoptotic role in CHO cells by reducing the expression of several anti-apoptotic genes and its transient inhibition delayed both the activation of Caspase-3/7 and the loss of cell viability. The present study evaluates the effect of stable inhibition of mmu-miR-466h-5p in CHO cells on their ability to resist apoptosis onset and their production properties. The expression of mmu-miR-466h-5p in the engineered anti-miR-466h CHO cell line was significantly lower than in the negative control and the parental CHO cells. These engineered cells reached higher maximum viable cell density and extended viability compared with negative control and parental CHO cells in batch cell cultures which resulted in the 53.8% and 41.6% increase of integral viable cells. The extended viability of anti-miR-466h CHO cells was the result of delayed Caspase-3/7 activation by more than 35h, and the increased levels of its anti-apoptotic gene targets (smo, stat5a, dad1, birc6, and bcl2l2) to between 2.1- and 12.5-fold compared with the negative control CHO in apoptotic conditions. The expression of secreted alkaline phosphatase (SEAP) increased 43% and the cell-specific productivity increased 11% in the stable pools of anti-miR-466h CHO compared with the stable pools of negative control CHO cells. The above results demonstrate the potential of this novel approach to create more productive cell lines through stable manipulation of specific miRNA expression.

Figure 1

Relative expression of mmu-miR-466h-5p in single CHO cell clones after PBA treatment. Single colonies of parental CHO and CHO transfected with negative control, anti-pre-miR-466h and anti-miR-466h-5p shRNAs were treated with PBA to compare activation of mmu-miR-466h-5p TaqMan qRT-PCR assays were used for analysis with snoRNA202 used as control for 2^-ΔΔCt analysis. The levels of mmu-miR-466h-5p in each clone after PBA treatment were related to its levels in the same clone treated with solvent.

Figure 2

Comparison of growth and viability of anti-miR-466h, negative control and parental CHO cells in batch cell culture. (A) Growth. (B) Viability.

Figure 3

Activation of Caspase-3/7 in anti-miR-466h and negative control CHO cells. (A) Time course of Caspase-3/7 activation in both cell lines (B), (C) and (D) FACS images of the ratio of viable cells negative for Caspase-3/7 activity (shown in red) and viable cells positive for Caspase-3/7 activity (shown in blue). (B) Negative control CHO at 236h. (C) Anti-miR-466h CHO at 236h. (D) Anti-miR-466h CHO at 270h.

Figure 4

Relative levels of mmu-miR-466h-5p in anti-miR-466h and negative control CHO cells at selected time points. The levels of mmu-miR-466h-5p before apoptosis onset (192 h) and after apoptosis onset (216, 226 and 236h) were related to their respective levels after 24 hours. TaqMan microRNA qRT-PCR analysis was used to assess mmu-miR-466h-5p levels with snoRNA 202 as control for 2^-ΔΔCt analysis.

Figure 5

Relative expression of the mmu-miR-466h-5p target genes in anti-miR-466h and negative control CHO cells at selected time points. The relative changes in genes expression are compared at 192, 216, 226 and 236h. (A) smo, (B) stat5a, (C) dad1, (D) birc6, (E) bcl2l2. TaqMan qRT-PCR assays were used for analysis with 18S used as control for 2^-ΔΔCt analysis.

Figure 6

SEAP activity and growth of SEAP-expressing anti-miR-466h and negative control CHO stable pools. Anti-miR-466h and negative control CHO cells were stably transfected with SEAP and stable pools were grown in batch conditions (A) SEAP activity in the culture media, (B) Cell growth