| Background and objectiveGlaucoma causes permanent optic nerve damage, which damages the visual function seriously. So glaucoma leads to irreversible blindness and the incidence rate is the highest in the world. Pathological features of glaucoma are pathologically elevated intraocular pressure and optic nerve atrophy, which lead to the occurrence of progressive visual field defect. But the pathogenesis of glaucoma has not been elucidated. Now mechanical compression damage theory and ischemia theory are considered to be related to its pathogenesis. The important risk factors in the pathogenesis of glaucoma is the intraocular hypertension. The main treatment for glaucoma is the use of medication or operation to reduce intraocular pressure, so as to slow the progression of glaucoma development. In clinical, the intraocular pressure can be reduced to normal in part of patients with glaucoma, but progress of optic atrophy cannot be blocked and visual function continues to decline. So the most important treatment is to application of protective drugs or other intervention to protect optic nerves. To prevent apoptosis fo optic ganglion cells and increase the number of surviving retinal ganglion cells and prolong the survival time are the key methods to keep the VA and preventing the damage of visual function of glaucoma patients.The formation of the optic nerve is mainly composed of retinal ganglion cells (RGCs) from the retina in all directions extend to the optic papilla and axonal fibers. Retinal ganglion cells of optic nerve transmit visual signals to the vision centre; therefore RGCs apoptosis is one of the important pathological features of glaucoma and other diseases in ophthalmology. The pathogenesis study of optic nerve damage disease mainly focuses on RGCs cells. To explore the mechanism of apoptosis of retinal ganglion cells, deprivation of neurotrophic factor, excitotoxicity, calcium overload, and the oxygen free radical injury can be used to prepare for RGC-5 apoptosis model. And the establishment of RGC-5 cell line and preparation of related apoptosis model is the key to study the damage mechanism of retinal ganglion cells.MicroRNA (miRNA), the highly conserved gene regulatory factor, provides new ideas for the treatment and diagnosis of diseases. miRNA is in a specific sequence manner and plays a role in post transcriptional regulation, which regulate gene expression on the level of inhibition of post transcriptional gene expression. MicroRNAs are involved in the regulation of cell growth, proliferation, differentiation, metabolism and apoptosis. miRNA consists of 19-25 nucleotides, 18-22 nt length. And precursor RNA by enzyme cut into small Drosha hairpin precursors of about 70 nt. miRNAs play on the degradation of target mRNA or the post transcriptional repressor function of translation. The study found that the microRNA is closely related to the occurrence and development of retinopathy. MiRNAs might be associated with optic nerve hemangioblastoma, retinitis pigmentosa, and retinal neovascularization etc. microRNA-100 (miR-100) is the new member of microRNAs. At present, miR-100 is considered as the main factor related to tumorigenesis, development of tumors. The abnormal expression of miR-100 has been found in liver cancer, lung cancer, nasopharyngeal carcinoma, adrenal cortical tumor and so on. The abnormal expression of miR-100 can promote the occurrence and invasion of tumors. But the expression of miR-100 in glaucoma and other ophthalmology diseases and its mechanism has not been completely elucidated. Therefore, this study aims to investigate the effect and mechanism of miR-100 on RGC-5 cell apoptosis.Methods:1.RGC-5 cells were cultured with different concentration (100 μm、200μm、400 μm、500 μm、1000 μm) hydrogen peroxide for 24h to make oxidative stress damage model. MTT and TUNEL method was used to detect the apoptosis of RGC-5 cells. Expression of miR-100 was detected by Real-time PCR in normal RGC-5 cells and different concentrations of hydrogen peroxide oxidative stress of the damage model.2. Using lentiviral transfection of miR-100 inhibitors is used to detect the apoptosis of RGC-5 cells. Effect on axon length by oxidative stress injury was detected by immunohistochemical analysis after inhibition of miR-100.3. Western blot analysis was employed to study the mechanism of miR-100 on RGC-5 cells. Luciferase activity was used to examine the targeted regulation of miR-100 and GIF 1R gene 3’UTR. After transfection of siRNA IGF1R, Western blot analysis and TUNEL assay were employed to detect RGC-5 cell apoptosis and its mechanism.Results:1. Oxidative stress damage caused by H2O2 increased the apoptosis rate of RGC-5 cells significantly, with significant difference compared to the control group (P<0.05). Apoptosis rate of RGC-5 cells caused by oxidative stress injury in a concentration dependent manner. In both normal RGC-5 cells and oxidative stress injury cell model, the expression of miR-100 could be detected. But in normal RGC-5 cells, miR-100 content is very low, and in oxidative stress injury cell model, miR-100 increased obviously, with statistically significant differences compared to the control group (P<0.05), and with a concentration dependent manner.2. After transfection of miR-100 inhibitor, the expression of miR-100 can significantly reduce in oxidative stress damaged RGC-5 cells, with significant difference compared with the negative control group (P<0.05). The expression of miR-100 in RGC-5 cells was effectively inhibited after transfection of miR-100 inhibitor in oxidative stress, and TUNEL positive cells were significantly reduced also, apoptosis rate of RGC-5 cells decreased obviously, compared with the negative control group, with significant difference (P<0.05). After transfection of miR-100 inhibitor, cells caused by oxidative stress injury became slender, morphology, which were similar to normal RGC-5 cells. RGC-5 cell neurite length was significantly increased, compared with the negative control group, with significant difference (P<0.05).3.After transfection of miR-100 inhibitor, the expression of phosphorylated TrkB, phosphorylated AKT, phosphorylated ERK1/2 protein increased, compared with the negative control group, with significant difference (P<0.05). MiR-100 targets IGF1R combinated of 3’UTR, luciferase activity of IGF1R 3’UTR decreased, and the decreased activity was mainly in the wild-type IGF1R. Compared with the blank control group and mutant IGFIR, the changes were with statistically significant difference (P<0.05). SiRNA interference of IGFIR, the apoptosis of RGC-5 reduced and TUNEL positive cells decreased significantly. The phosphorylation of AKT increased, with significant difference compared with the negative control.Conclusions:1 Hydrogen peroxide results in oxidative stress injury in RGC-5 cells and can induce the apoptosis of RGC-5 cells with a dose-dependent manner.2 MiR-100 was expressed in normal and oxidative stress injury RGC-5 cells; and with the oxidative stress injury degree increased; the expression of miR-100 was in a concentration dependent manner, suggesting that RGC-5 cell apoptosis is associated with miR-100.3 Downregulation of miR-100 can inhibit the apoptosis of RGC-5 cells and plays a protective effect on RGC-5 cells which may be through the activation of the TrkB/AKT/ERK pathway.4 Downregulation of the expression of miR-100 can regulate the expression of its target gene IGF1R, and result in activating of the TrkB/AKT/ERK pathway amd decrease RGC-5 cell apoptosis after oxidative stress which exerting a protective effect on RGC-5 cells. |
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