title: A novel microRNA mmu-miR-466h affects apoptosis regulation in mammalian cells.
abstract: This study determined the changes in microRNA (miRs) expression in mammalian Chinese hamster ovary (CHO) cells undergoing apoptosis induced by exposing the cells to nutrient-depleted media. The apoptosis onset was confirmed by reduced cell viability and Caspase-3/7 activation. Microarray comparison of known mouse and rat miRs in CHO cells exposed to fresh or depleted media revealed up-regulation of the mouse miR-297-669 cluster in CHO cells subjected to depleted media. The mmu-miR-466h was chosen for further analysis as the member of this cluster with the highest overexpression and its up-regulation in depleted media was confirmed with qRT-PCR. Since miRs 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 was used to predict and verify mmu-miR-466h anti-apoptotic targets. 8708 predicted targets were obtained from miRecords database and narrowed to 38 anti-apoptotic genes with DAVID NCBI annotation tool. 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. The qRT-PCR analysis revealed reduced mRNA levels of bcl2l2, dad1, birc6, stat5a, and smo genes in CHO cells exposed to depleted media. The inhibition of the mmu-miR-466h increased the expression levels of those genes and resulted in increased cell viability and decreased Caspase-3/7 activation. The up-regulation of mmu-miR-466h in response to nutrients depletion causes the inhibition of several anti-apoptotic genes in unison. This suggests the pro-apoptotic role of mmu-miR-466h and its capability to modulate the apoptotic pathway in mammalian cells.
authors.affiliation: Biotechnology Core Laboratory National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike Bldg 14A Rm 176, Bethesda, Maryland 20892, USA.
authors.forename: Aliaksandr
authors.initials: A
authors.lastname: Druz
authors.forename: Chia
authors.initials: C
authors.lastname: Chu
authors.forename: Brian
authors.initials: B
authors.lastname: Majors
authors.forename: Rodell
authors.initials: R
authors.lastname: Santuary
authors.forename: Michael
authors.initials: M
authors.lastname: Betenbaugh
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
mesh_terms: D000818:Animals; D017209:Apoptosis; D016466:CHO Cells; D053148:Caspase 3 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000737:chemistry; D005502:Food; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D051379:Mice; D035683:MicroRNAs / Q000378:metabolism*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D003470:Culture Media; C555937:MIRN191 microRNA, mouse; D035683:MicroRNAs; D053148:Caspase 3; D053179:Caspase 7