Study On The Mechanism Of P-A Gene In Trabecular Meshwork Cells

Objective:To study the effects of P-A gene mutation on endoplasmic reticulum stress and autophagy related gene expression in trabecular reticulum cells,explore the mechanism of endoplasmic reticulum stress and autophagy regulation induced by P-A gene mutation,and provide experimental evidence for the role of P-A gene mutation in primary congenital glaucoma.Methods:Human Trabecular meshwork cells were used as experimental objects,and the mutant plasmids of P-A gene were designed and constructed.The wild type and mutant plasmid of P-A gene(P-A-pc MV6-C.580 g >A)were transfected into trabecular reticulum cells in vitro to analyze the effect of P-A gene mutation on trabecular reticulum cells.Using RT-qPCR and Western Blot to detect ER stress-related genes,such as Glucose regulated protein78(GRP78)and Activating transcription factor6(ATF6),Inositolrequiring enzyme-1(IRE1),Protein kinase R-like endoplasmic reticulum kinase(PERK)and the mRNA and protein expressions of autophagy related genes(Beclin1,p62).After treatment with Endoplasmic reticulum stress inhibitor 4-phenylbutyric acid(4PBA)and autophagy inhibitor 3-methyladenine(3MA),Trabecular meshwork cells were transfected with mutant P-A gene to detect the expression of Endoplasmic reticulum stress and autophagy related genes.Finally,P-A-PCMV6 siRNA and GRP78-PCMV6 siRNA silence-related genes were screened to detect the expression of endoplasmic reticulum stress and autophagy related gene proteins.Results:The results of RT-qPCR and Western blot showed that P-A gene mutation significantly up-regulated the expression of endoplasmic reticulum stress-related genes(GRP78,ATF6,IRE1,PERK)and autophagy related genes(Beclin1,p62).Compared with the control group,the expression levels of endoplasmic reticulum stress-related genes(ATF6,IRE1,PERK)and autophagy related genes(Beclin1,p62)in the inhibitor preconditioning group were down-regulated.After silencing related genes,compared with the control group,the protein expression of ER stress and autophagy related genes decreased in the P-A gene silencing group.The expression of endoplasmic reticulum stress and autophagy related genes in the silenced GRP78 genome decreased compared with the control group.Conclusion:Endoplasmic reticulum stress of trabecular reticulum cells induced by P-A gene mutation may regulate autophagy through ATF6,IRE1 and PERK pathways.