Experimental Study Of Dickkopf-1 Inhibits Proliferation Of Human Lens Epithelial Cells And Its Mechanism

Objective:To investigate the effect of DKK1 on the proliferation of human lens epithelial cells?LECs?through Wnt/?-catenin signaling pathway and its possible mechanism,so as to provide a new target for clinical treatment of posterior capsular opacification?PCO?.Methods:Human LECs line SRA 01/04 cells were divided into three groups:Wnt3a overexpression group,DKK1 group and control group.In the Wnt3a overexpression group,the Wnt3a gene expression vector was transfected into human LECs SRA 01/04 cells by liposome mediated transfection,and the cell model of PCO was constructed.DKK1 group added 100 ng/mL DKK1 48 hours after transfection of Wnt3a gene expression vector,while the control group transfected pcDNA3-HA expression vector.Western blot was used to verify the expression of the vector in the transfected cells;CCK-8 assay was used to detect the effect of DKK1 on the proliferation of SRA 01/04 cells;immunofluorescence was used to detect the localization of?-catenin expression after overexpression of Wnt3a;immunocytochemistry was used to detect the expression of proliferating cell nuclear antigen?PCNA?after overexpression of Wnt3a;Western blot was used to detect the expression of Wnt3a.The effect of a overexpression on the expression of downstream signal molecules Cyclin D1 and C-myc in?-catenin.Results:Western blot analysis confirmed that the expression of Wnt3a protein in SRA 01/04cells increased significantly after transfection of pcDNA3-HA expression vector.CCK-8assay showed that there were significant differences in the average absorbance?A?value between control group,Wnt3a overexpression group and DKK1 group(F _group=10.91,P=0.003).Average absorbance?A?value of the two groups at different time points were also significantly different(F_time=6.041,P=0.025).The positive rate of PCNA protein expression in SRA 01/04 cells in DKK1 group was?14.2%+2.3%?,which was significantly lower than that in Wnt3a overexpression group?43.4%+5.4%?.The difference was statistically significant?P<0.05?.At 48 hours after transfection,?-catenin protein was concentrated in the nucleus and cytoplasm of Wnt3a overexpression group,but only in the cytoplasm of control group.In DKK1 group,?-catenin protein was mainly distributed in the cytoplasm and a small amount in the nucleus.Western blot analysis showed that the expression of Wnt/?-catenin signaling pathway target proteins Cyclin D1 and C-myc in DKK1 group was significantly lower than that in Wnt3a overexpression group.Conclusion:DKK1 inhibits Wnt/?-catenin signaling pathway activation induced by Wnt3a overexpression,down-regulates the expression of a subset of target genes,including Cyclin D1 and C-myc,and inhibits the proliferation of human LECs in SRA01/04 cells.