The miR-183/96/182Cluster Regulates Oxidative Apoptosis and Sensibility of Gliomas Cells to Chemotherapy
|School||Central South University|
|Course||Pathology and Pathophysiology|
|Keywords||Glioma micoRNA miR-183 miR-182 miR-96 AKT P53 Temozolomide Apoptosis Mitochondrialmembrane potential|
[Background]Many microRNAs reside in clusters in the genome. The miRNAs in the same cluster are generally similar in sequence, which are transcribed in the same direction, and usually function synergistically. MiR-183, miR-96and miR-182comprise the miR-183/96/182cluster. The members of the human miR-183/96/182cluster are located within4413bp of one another on human chromosome7q32.2and are transcribed in the same direction. These miRNAs are encoded in intergenic regions and have independent promoters. Furthermore, the coordinate expression of these miRNAs may function in physiology and pathology, e.g., in tumors. The human miR-183/96/182cluster is overexpressed in several tumors, and this overexpression is a good marker for bladder cancer, prostate cancer and urinary cytology. The pleiotropic effects of the miR-183/96/182cluster converge to regulate cell survival, proliferation and migration in medulloblastomas. However, the effects of their coordinate expression on mechanisms of tumorigenesis and the progression of gliomas are not well known.[MiR-183/96/182cluster regulate cell proliferation and apoptosis of glioma cell]Many apoptotic stimuli cause an early mitochondrial activation characterized by rapid induction of respiration-related proteins, which is then rapidly imported into the mitochondria to participate in the mitochondrial respiration, leading to early membrane hyperpolarization, increased oxygen consumption. Thus, we detected the effect of miR-183/96/182cluster on mitochondrial membrane potential (MMP,△Ψm) in U251cells by JC-1methods. We found that MMP is high and JC-1predominantly appears as red fluorescence in control cells (NEG), and knockdown miR-183cluster results reduced MMP and increased green fluorescence, green fluorescence of pooled knockdown of miR-183cluster was much more than that of individual component, and among these, miR-96knockdown achieves the most obvious effect on cell MMP.In situ hybridization analysis and real-time quantitative PCR analysis demonstrate that miR-183, miR-96and miR-182were expressed at relatively high levels in glioma tissue. We performed MTT analysis after transfected individual or all components of the miR-183/96/182cluster in U251cells and found that the overexpression of miR-183/96/182cluster promoted the proliferation of glioma cells. We also knocked down individual or all components of the miR-183/96/182cluster in U251cells, and then stained using Hoechst to detect the apoptosis. We noted an overall increase in cell apoptosis upon knockdown of the miR-183/96/182cluster. Therefore, overexpressing miR-183/96/182cluster can promote cell proliferation, while cell apoptosis is promoted when miR-183/96/182cluster are knocked down.[FGF9, CPEB1and FOXO1are the target gene of miR-183cluster]FOXO1is a known common target of miR-183cluster. We found FGF9and CPEB1are also predicted targets of mature miR-183,96and182using online software, and luciferase assay demonstrated the mature miR-183,96and182can combine with the3’-UTR area of FGF9or CPEB1, respectively. Extracted total RNAs and protein after transfected individual or all components of the miR-183/96/182cluster in U251cells, and then analyzed by qRT-PCR and western blot. We found that the expression of FGF9, CPEB1and FOXO1is increased when miR-183/96/182cluster is overexpressed. However, knocking down individual or all components of the miR-183/96/182cluster increased the expression of FGF9, CPEB1and FOXO1.[The effect of the miR-183/96/182cluster on intracellular reactive oxygen species production through their target gene FGF9, CPEB1and FOXO1]Intracellular reactive oxygen species (ROS) production was measured by counting the number of cells that were stained with carboxy-H2DCFDA using a spectrofluorometer after overexpressed or knocked down the miR-183/96/182cluster for48hours. We noted that the overexpression of the miR-183/96/182cluster resulted in the decrease of intracellular ROS production, but the production of intracellular ROS are increased when knocking down the miR-183/96/182cluster. Knocked down the miR-183/96/182cluster and following treated with NAC (N-acetyl cysteine, a ROS inhibitor that can inhibit intracellular ROS production), and then analyzed the intracellular ROS production using the same method. We found a decrease of intracellular ROS production. Therefore, overexpress the miR-183/96/182cluster can inhibit the production of intracellular ROS, which is promoted when this cluster is knocked down, so that NAC treatment significantly blocked the effects on ROS production from knocking down individual components of the miR-183cluster. Consistent with the pattern of mimic-183,96,182, interference with FGF9, CPEB1and FOXO1resulted in reduced ROS production and blocked the effect of miR-183,96and182on ROS increase [The effect of the miR-183/96/182cluster on the AKT/P53/apoptosissignaling pathway] Two of the most powerful signal transduction pathways activated byROS are those involving AKT and P53signaling. ROS signaling appears to be triggered by the activation of the mitochondrial dependent cell death pathway through AKT pathways, whereas p53mediates ROS-induced antiproliferative actions and early senescence or apoptosis. To investigate the effect of the miR-183/96/182cluster on the AKT/p53/apoptosis signaling pathway, mitochondria-related apoptosis molecules in U87cells with different treatment were examined by western blot analysis. Overexpression of the miR-183cluster significantly led to an increase expression of p-AKT, Bcl-2, and a decrease expression of Bax, P53. Meanwhile, Overexpression of the miR-183cluster caused an increase in the expression of mitochondrial cytochrome c and caspase9, which caused a concomitant decrease in the expression of cytosolic cytochrome c and caspase9. However, the pooled knockdown of the miR-183cluster resulted in opposite effects from overexpression of the pooled miR-183cluster.Consistent with the pattern of mimic-183,96,182, interference with FGF9, CPEB1and FOXO1resulted in a decrease of P53, while p-AKT is not. Therefore, miR-183cluster-induced AKT phosphorylation is independent of the target genes FGF9, CPEB1and FOXO1, but miR-183cluster-induced decreased expression of p53is dependent on those three genes. Consequently, inhibition of miR-183/96/182cluster induced ROS-mediated AKT/apoptosis signaling independent of three target genes FGF9, CPEB1and FOXO1, whereas, deletion of the clusters induced ROS-mediated P53/apoptosis signaling dependent of three target genes FGF9, CPEB1and FOXO1[Knockdown of the miR-183/96/182cluster sensitizes cells to chemotherapy in gliomas]To investigate whether knockdown of the miR-183/96/182cluster could also affect the pharmacological inhibition of TMZ, U87cells were used to evaluate the anti-tumor effects of the deigned treatments in vitro. TMZ can increase the production of intracellular ROS and the rate of apoptosis cells, and knockdown of miR-183/96/182cluster caused a more significant change compared to TMZ treatment only. Therefore, knockdown of the miR-183cluster sensitizes glioma cells to TMZ.Collectively, these results suggest that the miR-183/96/182cluster centralizes to participate in ROS-induced oxidative apoptosis and sensitizes cells to chemotherapy in gliomas.