MiR-184 Prevents Chronic Oxidative Stress Induced Human Trabecular Meshwork Cells Apoptosis And Cytotoxicity In Vitro By Targeting Hypoxia-inducible Factor 1?

Glaucoma which is due to increased intraocular pressure caused by the optic disc?formerly known as the optic nerve?depression,visual field defects,and ultimately can lead to blind eye disease.Normal eye pressure of 10?21 mmHg?Schitz tonometer?,more than 24mmHg for the pathological phenomenon.Increased intraocular pressure can lead to visual impairment,discs appear large and deep depression,visual field can be seen typical changes in glaucoma.The longer the intraocular pressure increases,the more severe the visual impairment.Glaucoma increased intraocular pressure is due to the dynamic balance of aqueous humor circulation has been destroyed.A small number due to excessive secretion of aqueous humor,but most of the outflow of aqueous humor or obstacles,such as the anterior chamber angle narrow or even closed,trabecular hardening.Glaucoma is a more common type of visual disorder.Glaucoma is a class of visual deterioration caused by optic nerve disease,optic nerve structure degradation,and then lead to optic nerve function loss.Glaucoma is often accompanied by symptoms of oxygen stress,apoptosis of retinal ganglion cells,changes in extracellular matrix components,optic atrophy,and visual loss due to elevated intraocular pressure.The decrease of the structure of the trabecular meshwork will lead to the increase of the intraocular pressure.When the trabecular meshwork is increased,the decrease of the intraocular pressure is favorable,which indicates that the trabecular meshworking plays an important role in regulating the intraocular pressure to prevent the optic nerve injury effect.Human trabecular cells are widely used as models of in vitro glaucoma disease.At present,more and more research show that reactive oxygen stress-induced oxygen stress is a phenomenon prevalent in neurodegenerative diseases,and oxygen stress response plays an important role in the development of glaucoma.Oxygen stress response caused by mitochondrial DNA damage and retinal pigment cells within the regulation of gene expression disorders,can lead to age,including macular degeneration,including a variety of optic neuropathy.MicroRNAs are small?18-25 nt?,endogenous noncoding oligoribonucleotides that are highly conserved across species and modulate the posttranscriptional silencing of gene expression.21 They function through recognition of specific sequences in their target mRNAs and predominantly act to reduce expression of their mRNA targets.22Understanding how these molecules modulate the mechanisms associated with glaucomatous damage may facilitate future targeting of microRNAs to attenuate glaucomatous injury.The crucial regulatory roles of miRNAs in the biology of glaucoma had been identified,suchas miR-29 family,miR-204,miR-24,and miR-184.miR-184 was first discovered by Lagos-Quintana et al in the mouse heart and cerebellum,miR-184 has a significant regulatory role in angiogenesis in ischemic disease by inhibiting VEGF and IGF-2 expression by binding to its target VEGF and IGF-2,thereby reducing angiogenesis.MiR-184acts as a potential oncogenic miRNA for humancancers,such as squamous cell carcinoma of tongue,malignant gliomas.Studies have shown that miR-184 can regulate angiogenesis by regulating Akt signaling pathway,TNF-a signaling pathway,and VEGF.It has been found that miR-184 expression levels are reduced in many patients with vision disorders.The expression of miR-184 in the cornea and trabecular meshwork in patients with keratoconus was higher than that in the ciliary body and retinal tissue,suggesting that miR-184 may play an important role in the prevention of optic neuropathy.Oxidative stress injury causes apoptosis and induces damage to the optic nerve tissue.It is one of the major causes of glaucoma,cataract and age-related macular degeneration.We investigated the molecular mechanism of miR-184 regulating the growth,apoptosis and oxidative stress-induced cytotoxicity of human trabecular meshwork cells by culturing human trabecular meshwork cells in vitro and simulating the oxidative stress injury.Target to provide a theoretical basis.Part I The expressions of miR-184 levels in human trabecular meshwork cells under chronic oxidative stress[Purpose]To investigate the expression of miR-184 levels in human trabecular meshwork cells under chronic oxidative stress.[Methods]HTM cells were generated from cadaver eyes tissues and incubated at 40%02 and 5%CO2 for 5 days to induce COS stressed cell model.Cell apoptosis,cytotoxicity,and extracellular matrix?ECM?expressions were studied in cells transfected with miR-184 mimic,inhibitor,or controls.Statistical analysis;Each experiment was performed in triplicate.All the quantitative data were expressed as mean value ±standard deviation?SD?in three independent experiments.Statistical analysis was performed using SPSS 19.0?SPSS Inc.,USA?,and statistical graphs were made using Graphpad Prism 6.0?GraphPad Software Inc,La Jolla,CA?.Differences between groups were analyzed by Tukey's test,and among three groups and more than three groups were analyzed using ANOVA,respectively.P<0.05 was considered as the statistically significant.[Results]1.There were no significant differences in the morphology of human trabecular meshwork cells under normal culture conditions and under chronic oxygen stress.2.The level of miR-184 in human trabecular meshwork cells under chronic oxygen stress was significantly lower than that in human trabecular meshwork cells under normal culture conditions?p<0.05?.3.Under chronic oxygen stress conditions,overexpressing miR-184 mimics,compared with the negative control group,the expression level of miR-184 in human trabecular meshwork cells increased significantly?p<0.05?;The expression of miR-184 in human trabecular meshwork cells was significantly lower than that in the negative control group after miR-184 inhibitors were transfected?p<0.05?.[Conclusion]Under the condition of chronic oxygen stress,miR-184 level was significantly decreased in human trabecular meshwork cells,and miR-184 had important regulation effect on human trabecular meshwork cells.Part ? The effect of miR-184 on the growth,apoptosis and cytotoxicity of human trabecular meshwork cells under oxidative stress[Purpose]To investigate the effects of miR-184 on cell growth,apoptosis and cytotoxicity of human trabecular meshwork under oxidative stress.[Methods]HTM cells were generated from cadaver eyes tissues and incubated at 40%O2 and 5%CO2 for 5 days to induce COS stressed cell model.Cell apoptosis,cytotoxicity,and extracellular matrix?ECM?changes were studied in cells transfected with miR-184 mimic,inhibitor,silenced?si-?HIF-1?,or controls.Use mimic of mi-184 and miR-184 inhibitor inquiry on the human trabecular meshwork cells apoptosis,cell toxicity and the effect of extracellular matrix components.[Results]1.Under the condition of chronic oxidative stress in two human trabecular meshwork cell lines,the cytotoxicity of human trabecular meshwork cells was significantly increased compared with that under normal conditions?p<0.05?.The increase in cytotoxicity caused by chronic oxidative stress conditions was significantly inhibited by the simulants expressing miR-184 in trabecular meshwork cells?p<0.05?.The cytotoxicity of human trabecular meshwork cells were significantly improved by the expression of miR-184 in human trabecular meshwork cells?p<0.05?.2.Under the conditions of chronic oxidative stress in the two human trabecular meshwork cell lines,the early apoptotic levels of the cells were significantly increased compared with those of the human trabecular meshwork cells under normal conditions?p<0.05?;the increase in early apoptosis levels caused by chronic oxidative stress conditions were inhibited significantly by the simulants expressing miR-184 in trabecular meshwork cells?p<0.05?;the early apoptosis of human trabecular meshwork cells can be improved significantly by the expression of miR-184 in human trabecular meshwork cells?p<0.05?.3.apoptosis-related protein Bax protein,shear type of Caspase-3 protein and mRNA expression levels were significantly decreased when miR-184 mimetic agents overexpressed?p<0.05?.apoptosis-related protein Bax protein,shear type of Caspase-3 protein and mRNA expression levels were significantly increased;when miR-184 inhibitors were transfected?p<0.05?.4.Under the condition of chronic oxidative stress in human trabecular meshwork cells,compared with the human trabecular meshwork cells under normal conditions,the mRNA and protein levels of COL1A1 protein,COL1A1 protein,COL3A1 protein,COL5A1 protein and SPARC protein of extracellular matrix protein were significantly increased?P<0.05=.the increase in extracellular matrix protein?COL1A1 protein,COL1A1 protein,COL1A1 protein,COL3A1 protein,COL5A1 protein and SPARC protein?expression levels caused by chronic oxidative stress conditions were inhibited significantly by the simulants expressing miR-184 in trabecular meshwork cells.Expression of miR-184 in human trabecular meshwork cells,the expression of extracellular matrix protein?COL1A1 protein,COL1A1 protein,COL1A1 protein,COL3A1 protein,COL5A1 protein and SPARC protein?in human trabecular meshwork cells can be improved significantly?P<0.05?.[Conclusion]Part III miR-184 regulates the growth,apoptosis and cytotoxicity of human trabecular cells under oxidative stress by acting on HIFla[Purpose]To investigate the effect of miR-184 on the apoptosis,toxicity and expression of foreign matrix proteins in human trabecular meshwork cells by regulating HIF-1 a under oxidative stress.[Methods]HTM cells were generated from cadaver eyes tissues and incubated at 40%02 and 5%C02 for 5 days to induce COS stressed cell model.Cell apoptosis,cytotoxicity,and extracellular matrix?ECM?changes were studied in cells transfected with miR-184 mimic,inhibitor,silenced?si-?HIF-1?,or controls.Use mimic of miR-184 and miR-184 inhibitor inquiry on the human trabecular meshwork cells apoptosis,cell toxicity and the effect of extracellular matrix components.We found that miR-184 could be directly by combining with the implementation of HIF1 alpha promoter regions of the inhibition of alpha,and through HIF1 alpha level achieved on the human trabecular meshwork cells apoptosis,cell toxicity and extracellular matrix components change control.[Results]1.The 3'UTR binding site of miR-184 binding to HIF1-a mRNA was predicted by http://www.targetscan.org/vert₇1/?.2.After transfection of miR-184 mimetic,the fluorescence activity of HIF-la 3'UTR was inhibit significantly?p<0.05?.It suggests that miR-184 can bind to 3'UTR of HIF-1a to inhibit the expression of HIFla.After mutating the predicted binding site and transfection of miR-184 mimetic agent,there was no significant effect on the activity of HIF-1la 3'UTR which suggested that the HIF-1a 3,UTR site of miR-184 binding is a previously predicted site.3.The expression of HIF-la mRNA and protein expression levels were inhibit significantly?p<0.05?after miR-184 simulants were transfected into human trabecular meshwork cells,and the expression of HIF-1a mRNA and protein expression levels were improved significantly?p<0.05?.4.After HIF-la lentivirus knockdown plasmid was transfected into human trabecular meshwork cells,immunofluorescence and immunoassay results showed that the expression level of HIF-1a was significantly decreased.5.Under oxidative stress conditions,when transfected with miR-184 inhibitors,the increase in cytotoxicity of human trabecular meshwork cells caused by oxidative stress were significantly promoted while the HIF-la lentivirus knockdown plasmid was transfected at the same time,the cytotoxicity of human trabecular meshwork cells decreased significantly.6.Under oxidative stress conditions,when transfected with miR-184 inhibitors,the rise of apoptosis of human trabecular meshwork cells caused by oxidative stress were significantly promoted,while the HIF-1a lentivirus knockdown plasmid was transfected at the same time,the apoptosis of human trabecular meshwork cells decreased significantly.7.Under oxidative stress conditions,when transfected with miR-184 inhibitors,the rise of extracellular matrix protein expression levels of human trabecular meshwork cells caused by oxidative stress were significantly increased while the HIF-1a lentivirus knockdown plasmid was transfected at the same time,the expression level of extracellular matrix protein in human trabecular meshwork cells decreased significantly.[Conclusion]Taken together,we concluded that miR-184 inhibited HTM cells cytotoxicity,apoptosis and ECM proteins expression via targeting HIF-1? in vivo.We suggest that miR-184 could be used as a potential target for glaucoma management.