title: 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.
authors: Biotechnology Core Laboratory NIDDK, NIH, Building 14A, Bethesda, MD 20892, USA.
authors: Aliaksandr
authors: A
authors: Druz
authors: Young-Jin
authors: YJ
authors: Son
authors: Michael
authors: M
authors: Betenbaugh
authors: Joseph
authors: J
authors: Shiloach
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D016466:CHO Cells; D053148:Caspase 3 / Q000096:biosynthesis / Q000235:genetics; D053179:Caspase 7 / Q000096:biosynthesis / Q000235:genetics; D002470:Cell Survival / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D020868:Gene Silencing; D051034:Inhibitor of Apoptosis Proteins / Q000096:biosynthesis / Q000235:genetics; D035683:MicroRNAs
chemical_list: D051034:Inhibitor of Apoptosis Proteins; D035683:MicroRNAs; C576133:Mirn466 microRNA, mouse; D053148:Caspase 3; D053179:Caspase 7
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