The Study On Extracellular Matrix Of Atg7-knockout HTM Cell Using CRISPR/Cas9 System And Effect Of Melatonin On Chloride Efflux In Porcine Ciliary Epithelium

Purpose:Glaucoma is a group of disease characterized by the progressive degeneration of optic nerve and loss of visual field. Primary Open Angle Glaucoma (POAG), the most prevalent form of the disease worldwide, is a late onset disorder often associated with an increase in intraocular pressure (IOP). If the dynamic equilibrium between aqueous humour secretion and outflow is broken, the elevation of IOP may subsequently lead to POAGTrabecular meshwork is a main outflow pathway, and human trabecular meshwork (HTM) cells maintain a balance of secretion and degeneration of extracellular matrix (ECM), in which autophagy plays an important role. POAG is an age-related disease, the activity of autophagy decreases with age. Autophagy gene 7 (Atg7) is a significant gene in the regulation of early stage of autophagy process. We established glaucoma model through oxidative stress by H2O2 and utilized CRISPR/Cas9 system to study if autophagy has association with pathogenesis of POAGThe ciliary epithelium (CE) is the site of aqueous humor secretion, more and more evidences have proved that it showed a circadian rhythm of aqueous flow regulation. Melatonin released by CE is possibly a crucial regulatory mediator. Ciliary epithelium cells may have the ability to alter autophagy associated signal pathways of HTM cell, indicating a correlation between aqueous humour inflow and outflow pathways. We used porcine eyes as animal model to study the influence of melatonin in aqueous humour dynamics. This information is fundamental to our understanding of the precise functional significance of melatonin and also for devising future therapeutic approaches in glaucoma therapy.Methods:1. Recombined plasmid pEP-KO with sgRNA of Atg7 was conducted by CRISPR/Cas9 system. After transfected into HTM cells, we get a stable Atg7 gene knock out HTM cell line. We also got control group which was transfected with blank plasmid pEP-KO.2. We established oxidative stress model with H2O2 on HTM, HTM-pEP-KO and HTM-Atg7-/- cells. The expressions level of FN and Col Ⅳ was detected by western blot analysis, and we also detected primary signal pathways. Fluorescence microscopy was used to observe LC3 expression at different conditions.3. Ussing-type (UT) chamber was used to observe the effects of melatonin and NFA on PD and transepithelial Isc across porcine ciliary epithelium. Elucidate the potential signaling pathway of melatonin by Elisa.Results:1. We successfully established Atg7 knock out HTM cell line using CRISPR/Cas9 system, with a low ratio of LC3-Ⅱ/LC3-Ⅰ and overdeposition of p62 protein. No obvious off-target effect was observed.2. Baseline expression of FN and Col IV in HTM-Atg7-/- cells were more than HTM and HTM-pEP-KO cells (p<0.001). After treated with 100μM and 300μM H2O2, Col IV and FN expression increased in the three groups (p<0.05).3. At baseline level, phosphorylation of Akt was activated more in HTM-Atg7-/- cells, while phosphorylation of extracellular regulated kinase (Erk)1/2 was suppressed compared with the other groups (p<0.001, p<0.05). No significant difference was observed in p38 pathway. After treated with H2O2, Erkl/2 and p38 pathways were activated in all three groups (p<0.001, p<0.05), but p-Erk level in HTM-Atg7-/- group was lower and p-p38 level was higher than the other groups (p<0.01, p<0.05), and no significant change was observed on p-Akt expression.4. The percentage of LC3 fluorescent staining area observed by fluorescence microscopy was higher in HTM and HTM-pEP-KO groups than HTM-Atg7-/- group (p<0.05). No significant difference was found after H2O2 stimulation.5. When added in NPE side only and NPE together with PE sides, 100μM melatonin elevated Isc across porcine ciliary epithelium by 45% and 53%, respectively (p<0.001, p<0.01). NFA converted this elevation significantly.6. Both 10 and 100μM melatonin promoted cAMP formation in ciliary epithelium. After 1 hour, the effect of 10 and 100μM melatonin were statistically significant. After 3 hours, only 10μM melatonin had a significant effect (p<0.05).Conclusion1. We established Atg7 knock out HTM cell line, with a great deficiency in autophagy function, but was not completely blocked. No off target effect was found.2. Overdeposition of ECM in HTM-Atg7-/- cells may lead to POAG. Oxidative stress mediated p38 signaling pathway resulted in the increase of ECM expression. HTM activated Erk1/2 mediated Atg7-dependent autophagy pathway to promote cell survival.3. Melatonin triggered net Cl- secretion through elevating intracellular cAMP in isolated NPE and PE cells, increasing the potential risk of aqueous humour formation.