MiR-107 expression in MCF7 cells after hypoxia (0.1 O2 for 16, 24 and 48 h) vs. normoxia (P = 0.04). * denotes p < 0.05 compared with parallel controls. Data represent normalized mean ?S.E (error bars) (n = 3). miRNA levels were analysed by q-RT PCR and normalised to U6 levels. Statistical significance established by Student's t-test. Dicer expression PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28380356 was examined after transient transfection with miR-103/107 inhibitors and exposure to hypoxia vs. normoxia. C, Dicer protein expression after transfection of MCF7 cells with miR-103/107 inhibitors or control inhibitors and exposure to hypoxia (0.1 O2 for 48 h) vs. normoxia. Results show three technical replicates per treatment. Dicer and -actinin protein levels were examined by immunoblotting. -actinin was used as the loading control.In a similar experiment undertaken with a longer duration of hypoxic exposure (0.1 O2 for 48 h) several miRNAs were significantly up or down regulated in MCF7 cells (see Additional file 1: Figure S1). Eight miRNAs were significantly up regulated (see Additional file 1: Table S2), and four miRNAs were significantly down regulated in hypoxia when compared to normoxia (see Additional file 1: Table S3). Even following a longer duration of hypoxic exposure (0.1 O2 for 48 h) there were no significant changes between individual precursor miRNA: FPS-ZM1MedChemExpress FPS-ZM1 mature miRNA ratios either with hypoxia (see Additional file 1: Figure S2) or, surprisingly, following Dicer suppression by siRNA (see Additional file 1: Figure S3-S4). The microarray data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus [38] and are accessible through GEO Series accession number GSE49999.To explore the effects of hypoxia on the processing of specific miRNAs with a different assay, the levels of mature and precursor miRNA levels of let-7a, miR-21 and miR-185 were determined in MCF7 cells exposed to hypoxia vs. normoxia, by RT-PCR. These were selected as previous reports showed they were Dicer dependent miRNAs [44-46]. Only a modest decrease in mature and precursor levels of let-7a and miR-21 in hypoxia was observed, and no accumulation of pre-let-7a or pre-miR-21 in hypoxia was evident (Figure 10A, 10B). A significant reduction (P = 0.03) in mature miR-185 was observed in hypoxia in MCF7 cells (Figure 10C), but no accumulation of precursors was seen. Furthermore following the recent report of hypoxia reducing miRNA processing by Ho et al. [45] in HUVEC cells, the ratio of mature and pre-miRNA levels for miR-185 and miR-21 was examined in these cells.Bandara et al. BMC Cancer 2014, 14:533 http://www.biomedcentral.com/1471-2407/14/Page 11 ofFigure 8 Hypoxic regulation of other miRNA biogenesis proteins. Expression of miRNA biogenesis proteins Drosha, TARBP2, DGCR8 and XPO5 were examined under hypoxia vs. normoxia A, Drosha mRNA expression in SKBR3 cells after hypoxia (0.1 O2 48 h) vs. normoxia (P = 0.05). B, TARBP2 mRNA expression in SKBR3 cells after hypoxia (0.1 O2 48 h) vs. normoxia (P = 0.03). *denotes P < 0.05 compared with parallel controls. Data represent normalized mean ?S.E (error bars) (n = 3). mRNA levels were analysed by RT-PCR and normalised to 18S rRNA levels. C, Drosha, TARBP2, DGCR8 and XPO5 protein expression in SKBR3 cells after hypoxia (0.1 O2 48 h) vs. normoxia. Results show three technical replicates per treatment. Protein levels were examined by immunoblotting. -actinin and -tubulin used as the loading controls.However, following both 24 and 48 h o.
