Relationship Between The Altered Expression And Epigenetics Of GSTM3 And Age-related Cataract

Objective To observe the altered expression of glutathione S-Transferase Mu 3(GSTM3)in lens epithelial cells(LECs)and lens cortex of age-related cataract(ARC)and to examine the potential contribution of the promotor methylation and histone modification in GSTM3 to ARC pathogenesis.Methods A clinical case-control study was conducted in the department of ophthalmology from August 2014 to August 2015 in the Affiliated Hospital of Nantong University.The case group consisted of three subgroups: cortical cataract;nuclear cataract and posterior cataract.Each subgroup contained 40 eyes from 40 patients with no other ocular diseases according to the Lens Opacities Classification System III.Forty transparent control lens samples were obtained during vitrectomy.GSTM3 expression,DNA methylation and histone modification were assessed by quantificational real-time polymerase chain reaction(qRT-PCR),western blot,bisulfite-sequencing PCR(BSP),pyrosequencing and chromatin immunoprecipitation(Ch IP)assay.Human lens epithelial(HLE)cell lines,SRA01/04 and HLEB3,were chemically altered to observe the relationship between epigenetic status and expression of GSTM3.The cells were exposed to hydrogen peroxide(H2O2)for oxidative stress,demethylated by 5-Aza-2'-deoxycytidine(5-Aza-dC),inhibited of histone deacetylases by trichostatin A(TSA).Electrophoretic mobility shift assay(EMSA)was used to detect potential transcription factors binding to GSTM3 promoter.Statistical analyzes were performed with SPSS 17.0 software.Results The GSTM3 expression was significantly decreased in the ARC LECs and lens cortex than that of the controls(m RNA: F=308.90,P<0.01;F=198.42,P<0.01;protein: F=219.74,P<0.01;F=195.77,P<0.01),which corresponded to hypermethyaltion of the GSTM3 promoter(LECs of controls: 2.00±1.36,ARC-C: 20.11±1.23,ARC-N: 25.17±1.18,ARC-P: 17.94±1.51,F=1138.44,P<0.01;lens cortex of controls: 4.72±1.49,ARC-C: 19.80±1.26,ARC-N: 26.75±1.02,ARC-P: 20.03±1.36,F=1035.63,P<0.01).Lower GSTM3 was detected in HLEB3 cells than SRA01/04 cells(t=14.48,P<0.01;t=16.32,P<0.01).HLEB3 cells also displayed hypermethylation in comparison with SRA01/04 cells(SRA01/04: 1.25±0.67,HLEB3: 20.58±1.07,t=26.51,P<0.01),and acetylated H3 levels were lower while tri-methylated H3K9 increased in HLEB3 cells than SRA01/04 cells(t=19.21,P<0.01;t=6.16,P<0.01).After treatment with DNA methyltransferase inhibitor 5-Aza-dC or histone deacetylase inhibitor TSA,the GSTM3 expression increased in HLEB3 cells(t=7.24,P<0.01;t=10.94,P<0.01).Methylation of GSTM3 promoter abrogated the transcription factor binding.The expression of GSTM3 declined in H2O2-treated HLE cell lines(m RNA: F=54.43,P<0.01;F=66.82,P<0.01;protein: F=7.44,P<0.01,F=30.62,P<0.01),which corresponded to hypermethyaltion of the GSTM3 promoter(F=989.04,P<0.01;F=595.94,P<0.01).Conclusions A hypermethylation in GSTM3 promoter and altered histone modification in LECs and lens cortex might contribute to the ARC formation.