S119: MicroRNA regulation of apoptosis in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were induced to undergo apoptosis by exposing the cells to nutrient-depleted media. The apoptosis onset was confirmed by reduced cell viability and caspase-3/7 activation.  A microarray comparison of known microRNA's in CHO cells exposed to fresh or depleted media revealed up-regulation of the mouse miR-297-669 cluster in CHO cells when subjected to depleted media. Mmu-miR-466h was chosen for further analysis as the member of this cluster with the highest overexpression and its up-regulation was confirmed with qRT-PCR. Since microRNAs suppress mRNA translation, we hypothesized that up-regulated mmu-miR-466h inhibits anti-apoptotic genes and induces apoptosis. A combination of bioinformatics and experimental tools predicted 38 mmu-miR-466h anti-apoptotic targets. Several genes were selected from this anti-apoptotic subset based on nucleotide pairing complimentarity between the mmu-miR-466h seed region and 3' UTR of the target mRNAs. qRT-PCR analysis revealed reduced mRNA levels of bcl2l2, dad1, birc6, stat5a and smo genes in CHO cells exposed to depleted media.   Furthermore, the inhibition of the mmu-miR-466h increased the expression levels of these genes and resulted in increased cell viability and decreased caspase-3/7 activation. The inhibition of multiple anti-apoptotic genes suggests a pro-apoptotic role of mmu-miR-466h and its capability to modulate the apoptotic pathway in mammalian cell cultures.