The Role Of Wnt/β-catenin Signaling Pathway Regulated By ICAT In Monocytic Differentiation Of AML HL-60 Cells With 1,25-dihydroxyvitamin D3 Treatment)

Objective:To explore the role of ICAT with 1,25-（OH）₂D₃ treatment in HL-60 cells induced monocytic differentiation through Wnt/β-catenin signaling pathway.Methods:0.1μmol/L 1,25-（OH）₂D₃ was used to induce HL-60 cells to differentiate into monocytes,flow cytometry and cytochemical staining techniques were used to determine the differentiation direction and differentiation ratio of the cells;CCK-8 assay was used to determine the inhibition rate of cell proliferation;using flow cytometry to analyze the cell cycle of HL-60 cells.Using RT-q PCR and Western Blot to analyze the gene and protein expression of ICAT andβ-catenin in HL-60 cells before and after1,25-（OH）₂D₃ treatment,as well as the downstream target of Wnt signaling pathway（c-Myc、Cyclin D1 and TCF-1 protein）;using nuclear protein separation and identification technology to analyze the differences in the expression of ICAT andβ-catenin protein in the nucleus of HL-60 cells.At the same time,laser confocal technology（LSCM）was used to analyze the expression and localization of ICAT andβ-catenin proteins in cells.Subsequently,exogenous high expression of ICAT protein,by using RT-q PCR,Western Blot,flow cytometry and LSCM to analyze the the differentiation direction of HL-60 cells with high expression of ICAT,the p-β-catenin protein levels,nuclear translocation of ICAT andβ-catenin,and the expression of c-Myc,cyclin D1 and TCF-1 protein.Results:0.1μmol/L 1,25-（OH）₂D₃ could induce HL-60 cells differentiate into monocyte after 72 h.The proportion of CD14（+）of HL-60 cells reached[（96.93±3.84）%].It was significantly much more than that of the control group[（1.63±1.02）%]（P<0.01）.The result of Wright’s-Giemsa staining showed that the cell morphology was changed after 1,25-（OH）₂D₃ treatment.The nucleus was irregularly shaped,nucleolar disappeared.The ratio of nucleus and cytoplasm was obviously decreased.The cell cycle was arrest at G₀/G₁ phase with a percentage of[（65.2±5.3）%],higher than that of the control group[（49.4±2.2）%]（P<0.01）,CCK-8 assay suggested that 1,25-（OH）₂D₃ could significantly inhibit cell proliferation.The m RNA and protein expression of ICAT were significantly up-regulated（P<0.01）,β-catenin expression had no obvious change,butβ-catenin was down-regulated at the level of nucleoprotein（P<0.05）.p-β-catenin（ser33/37）protein expression level had no obvious change（P>0.05）,and Wnt/β-catenin signaling pathway’s target genes（c-Myc、cyclin D1 and TCF-1）were down-regulated（P<0.05）.Laser scanning confocal microscopy showed thatβ-catenin protein was decreased in nuclear and aggregated in the cytoplasm.After exogenous the expression of ICAT,the proportion of CD14（+）of ICAT-overexpressing cells[（48.55±1.8）%]was significantly higher than that of the control group[（2.23±0.34）%]（P<0.01）.β-catenin expression level of m RNA and total protein had no obvious change.At the same timeβ-catenin was down-regulated at the level of nucleoprotein,transferred into the cytoplasm（P<0.05）.In addition,p-β-catenin（ser33/37）protein expression level had no obvious change（P>0.05）,and Wnt/β-catenin signaling pathway’s target genes were down-regulated（P<0.01）.Conclusion:1,25-（OH）₂D₃ could up-regulating the expression of ICAT protein,inhibit HL-60 cell proliferation and induced the cell into monocytic differentiation through Wnt/β-catenin signaling pathway.