Regulatory effect of EZH2 upon GFRα1 promoter DNA methylation in SH-SY5Y cells
Objective To explore the molecular mechanism of EZH2 regulating GFRα1 promoter DNA methylation in SH-SY5Y cells.Methods After EZH2/DNMT3B overexpression and interference in SH-SY5Y cells,the expression levels of EZH2,DNMT3B and GFRα1 were detected by quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot.EZH2 binding level and H3K27me3 in DNMT3B promoter were detected by chromatin immunoprecipitation assay-PCR(ChIP-qPCR).DNA methylation of GFRα1 promoter in all transfected groups was detected by BSP.Independent sample t-test was utilized for comparing the mean of two groups.Multiple groups of data were examined by one-way ANOVA and LSD test.Results qRT-PCR and Western blot results showed that mRNA and protein relative expression levels of EZH2 in EZH2 overexpression group were higher than those in overexpression control group,and those in EZH2 interference group were lower than those in interference control group(all P＜0.05).The relative expression levels of mRNA and protein were higher in DNMT3B overexpression group than those in overexpression control group.The relative expression levels of DNMT3B mRNA and protein were lower in DNMT3B interference group than those in interference control group(all P＜0.05).The results of ChIP-qPCR showed that EZH2 binding level was higher in DNMT3B promoter region of EZH2 overexpression group than that of overexpression control group[(2.25±0.15)vs(1.00±0.05),t=-12.82,P=0.006].Methylation level of H3K27 was higher in DNMT3B promoter region of EZH2 overexpression group than that of overexpression control group[(2.47±0.44)vs(1.01±0.09),t=-6.17,P=0.025].EZH2 binding level of DNMT3B promoter in EZH2 interference group was lower than that in control group[(0.57±0.08)vs(1.00±0.06),t=5.80,P=0.029].The methylation level of H3K27 was lower in DNMT3B promoter of EZH2 interference group than that of control group[(0.31±0.09)vs(1.08±0.17),t=12.24,P=0.007].qRT-PCR and Western blot results showed that the relative expression level of DNMT3B mRNA in EZH2 overexpression group was lower than that in control group[(0.58±0.09)vs(1.01± 0.13),t=17.75,P=0.003].The relative protein expression level of DNMT3B was lower in EZH2 overexpression group than that in control group[(0.32±0.06)vs(0.54±0.05),t=23.64,P=0.002].The relative mRNA expression level of DNMT3B was higher in EZH2 interference group than that in control group[(1.79±0.05)vs(1.01±0.1),t=-12.00,P=0.007].The protein relative expression level of DNMT3B was higher in EZH2 interference group than that in EZH2 interference group[(0.85± 0.08)vs(0.51±0.07),t=-18.78,P=0.003].The results of bisulfite sequencing showed that DNA methylation level of GFRα1 promoter was lower in EZH2 overexpression group than that in overexpression control group[(0.28±0.03)vs(0.52±0.01),t=8.93,P=0.012].DNA methylation level of GFRα1 promoter was higher in EZH2 interference group than that in control group[(0.79± 0.02)vs(0.52±0.01),t=-44.45,P=0.001].qRT-PCR indicated that the relative expression of GFRα1 mRNA was higher in EZH2 overexpression group than that in control group[(1.87±0.05)vs(1.01±0.14),t=-10.04,P=0.001].The relative expression of GFRα1 mRNA was lower in EZH2 overexpression+DNMT3B overexpression group than that in overexpression control group[(0.73± 0.04)vs(1.01±0.14),t=3.27,P=0.031].The relative expression level of GFRα1 mRNA was lower in EZH2 overexpression+DNMT3B overexpression group than that in EZH2 overexpression group[(0.73±0.04)vs(1.87±0.05),t=30.00,P=0.001].Western blot revealed that the relative expression of GFRα1 protein was higher in EZH2 overexpression group than that in control group[(0.89±0.07)vs(0.59±0.03),t=-7.09,P=0.002].The relative expression of GFRα1 protein was lower in EZH2 overexpression+DNMT3B overexpression group than that in overexpression control group[(0.48±0.03)vs(0.59±0.03),t=3.51,P=0.025].The relative expression level of GFRα1 protein was lower in EZH2 overexpression+DNMT3B overexpression group than that in EZH2 overexpression group[(0.48±0.03)vs(0.89±0.07),t=8.84,P=0.001].qRT-PCR results showed that the relative expression of GFRα1 mRNA was lower in EZH2 interference group than that in control group[(0.57±0.13)vs(1.03±0.13),t=4.29,P=0.013].The relative expression of GFRα1 mRNA was higher in EZH2+DNMT3B interference group than that in control group[(1.59±0.06)vs(1.03±0.13),t=-6.67,P=0.003].The relative expression level of GFRα1 mRNA was higher in EZH2+DNMT3B interference group than that in EZH2 interference group[(1.59±0.06)vs(0.57± 0.13),t=-12.60,P=0.001].Western blot indicated that the relative protein expression of GFRα1 was lower in EZH2 interference group than that in control group[(0.28±0.05)vs(0.58±0.04),t=7.59,P=0.002].The relative expression of GFRα1 protein was higher in EZH2+DNMT3B interference group than that in control group[(0.79±0.07)vs(0.58±0.04),t=-4.16,P=0.014].The relative protein expression level of GFRα1 was higher in EZH2+DNMT3B interference group than that in EZH2 interference group[(0.79±0.07)vs(0.28±0.05),t=-9.78,P=0.001].Conclusions EZH2 may up-regulate the expression of GFRα1 through an inhibition of DNMT3B expression and a down-regulation of DNA methylation in promoter region of GFRα1 in SH-SY5Y cells.
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