Regulation and its mechanism of MeCP2 on biological behavior and epithelial-mesenchymal transition of LECs / 中华实验眼科杂志

ABSTRACT

Objective To investigate the role of methyl-CpG-binding protein 2 (MeCP2) in the regulation and epithelial-mesenchymal transition (EMT) of human lens epithelial cells (LECs) and its possible mechanism.Methods Human LEC lines (SRA01/04) were divided into MeCP2-mimic group,MeCP2-NC group and small interferening RNA-MeCP2 (si-MeCP2) group,and MeCP2 analog plasmid,blank plasmid and MeCP2 si-RNA plasmid was used respectively to transfect the cells.The expression of MeCP2 mRNA in the cells was detected by using real-time PCR 24 hours after transfection.At 48 hours after transfection,the migration rate of the cells was evaluated by scratching test,and the expression of Wnt3a protein in the cells was detected by immunofluorescence stainning.The relative expressions of β-catenin,E-cadherin,Vimentin,matrix metallo proteinase (MMP)-9,MMP-7 and secreted frizzled-related protein 5 (SFRP5) proteins in the cells were detected by Western blot.Results After 24 hours of transfection,the relative expression of MeCP2 mRNA in the cells was significantly different among the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group (F =4 773.00,P＜0.00 1).The migrating rate of the cells in the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was (57.45±5.20)％,(32.71± 10.02)％ and (17.77±9.22)％,respectively,showing a significant difference among the three groups (F=124.00,P＜0.001),and the migrating rate of the cells in the si-MeCP2 group was significantly lower than that of the MeCP2-mimic group or MeCP2-NC group (both at P＜0.001).The relative expressing intensity (absorbance) of Wnt3a in the cells of the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was 75.92 ± 6.10,52.03 ± 5.22 and 28.75 ± 3.39,respectively,with a significant difference among three the groups (F=221.30,P＜0.001),and the relative expressing intensity (absorbance) of Wnt3a in the cells was significantly lower in the si-MeCP2-mimic group than that of the MeCP2-NC group and MeCP2-mimic group (both at P＜0.001).The relative expressing level of E-cadherin protein was significantly elevated and the expressions of β-catenin,Vimentin,MMP-9 and MMP-7 were significantly reduced in the si-MeCP2 group compared with the MeCP2-mimnic group and MeCP2-NC group (all at P＜O.01).The relative expressing level of SFRP5 protein in the MeCP2-mimic group,MeCP2-NC group and si-MeCP2 group was 27.19± 0.03,47.54±0.05 and 74.93±0.05,respectively,showing a statistical difference among the three groups (F =183.49,P＜0.001),and the relative expressing level of SFRP5 in the si-MeCP2 group was significantly higher than that in the MeCP2-mimic group and MeCP2-NC group (both at P＜0.001).Conclusions MeCP2C can promote EMT of human LECs by down-regulating the expression of SFRP5 and therefore activating the Wnt3a/β-catenin signal pathway.