Supplementary Materialsoncotarget-07-12267-s001. manifestation during tumorigenesis are many and include, for example, haploinsufficiency by genomic rearrangements and altered gene dosage, epigenetic gene inactivation, and defects in Mad2 production at transcriptional or translational level. For example at the transcriptional level, overexpression of Mad2 protein has been observed upon adenovirus E1A -mediated inactivation of pRb, which causes stimulation of E2F-dependent transcription of Mad2 mRNA  while in cells with deregulated repressor-element-1-silencing transcription factor (REST) the Mad2 protein levels were found to be decreased . In both cases, the aberrant Mad2 levels were associated with mitotic defects causing aneuploidy. Here we report the discovery of a novel post-transcriptional regulator of Mad2, miR-493-3p, and demonstrate how excess of the miRNA causes aneuploidy and development of microtubule drug resistance in cancer cells. RESULTS Excess miR-493-3p compromises microtubule drug induced M phase arrest and in drug-free culture accelerates mitosis Mir-493-3p (Figure 1A-1B) was one of the hits from our cell-based high-throughput screen (HTS) for miRNAs that antagonize microtubule drug induced mitotic block . The majority of HeLa cervical cancer cells overexpressing miR-493-3p evaded mitotic block induced by a microtubule stabilizing drug taxol or microtubule depolymerizing agent nocodazole, and formed large progeny cells with a multilobed nuclear morphology (Figure ?(Figure1C).1C). This was in contrast to cells transfected with non-targeting control miRNA (miR-control), which exhibited a long mitotic arrest with condensed chromosomes when treated with the drugs (Figure ?(Figure1C).1C). To confirm the result and visualize the timing of forced mitotic exit by miR-493-3p we monitored taxol or nocodazole treated miR-control or miR-493-3p transfected cell populations using time-lapse microscopy. As expected, majority of the microtubule drug treated miR-control overexpressing cells arrested at M phase for longer than 8 hours before they underwent cell death (Figure ?(Figure1D).1D). In contrast, many cells with excess miR-493-3p exhibited a forced mitotic leave within 100 mins after admittance to M stage despite the existence of taxol or nocodazole in the culture medium (Physique ?(Figure1D).1D). Quantification of the time-lapse films indicated that in response to taxol, an average of MIF Antagonist 49.0 +/? MIF Antagonist 4.4% of the mitotic cells in the miR-493-3p overexpressing cell populations underwent the forced mitotic exit, which is significantly more compared to the average of 2.0 +/? 2.6% in the miR-control transfected controls (= 0.002, Figure ?Physique1D).1D). Comparable results were obtained with miRNA transfected and nocodazole treated cells (Physique ?(Figure1D)1D) as well as MIF Antagonist with synchronized HeLa cell populations that were released from G1/S block into growth medium containing taxol or nocodazole (Figure ?(Figure1E).1E). When cycling non-drug treated miR-control or miR-493-3p transfected HeLa cells were time-lapse filmed we MIF Antagonist noted a significant difference in the time the cells spent in mitosis (Physique ?(Figure1E);1E); the average time from nuclear envelope breakdown (NEBD) to onset of anaphase for the miR-control and miR-493-3p MIF Antagonist transfected cells was 35.7 +/? 1.7 min and 22.3 +/? 2.0 min, respectively (= 0.02). Based on these results we conclude that extra miR-493-3p enables cells to escape spindle poison induced M phase block and in drug-free culture conditions accelerates mitosis. Open in NFKB-p50 a separate window Physique 1 Excess of miR-493-3p weakens the SACA. Schematic illustration of the miR-493 hairpin loop. B. Quantification of the miR-493-3p levels in HeLa cells 24 h after the transfection with miR-control or pre-miR-493. C. Representative fluorescence pictures of DAPI stained nuclei of miR-control and miR-493-3p overexpressing HeLa cells set after right away treatment with taxol or nocodazole. Size club, 50m. D. Consultant still pictures of non-synchronized time-lapse filmed miR-control and miR-493-3p overexpressing HeLa cells treated with taxol or nocodazole (0 min = NEBD). Size club, 25 m. The graph displays quantification of compelled mitotic leave in miRNA transfected cell populations cultured in the current presence of taxol or nocodazole (= 300 cells per treatment). E. Quantification of mitotic indices of miRNA-transfected and synchronized HeLa cell populations after discharge through the thymidine stop into taxol or nocodazole (n600 cells examined for each period stage). F. Consultant still pictures of non-synchronized miRNA-transfected HeLa cells going through mitosis in drug-free lifestyle circumstances (0 min = NEBD). Size club, 25 m. The graph displays quantification of mitotic duration in the indicated miRNA transfected cells (= 300 cells per group). All data is certainly suggest +/? s.d. from 3 indie tests. The asterisks denote statistical significance (* = 0.05, ** = 0.01). miR-493-3p targets the 3UTR of Mad2 suppresses and mRNA Mad2 gene expression.