Effects of ICAT silencing in Wnt signaling pathway and NSC67657 induced cell differentiation of HL-60 cells
10.3760/cma.j.issn.0253-2727.2017.11.015
- VernacularTitle: HL-60细胞中ICAT基因沉默对Wnt信号通路及NSC67657诱导细胞分化的影响
- Author:
Weijia WANG
1
;
Kang CHEN
;
Juan WANG
;
Yunxiu HUANG
;
Decai ZHANG
Author Information
1. Zhongshan People’s Hospital, Guangzhou 528402, China
- Publication Type:Journal Article
- Keywords:
HL-60 cells;
β
-catenin interacting protein 1;
Differentiation;
Wnt signaling pathway
- From:
Chinese Journal of Hematology
2017;38(11):972-976
- CountryChina
- Language:Chinese
-
Abstract:
Objectives:To investigate the effect of β-catenin interacting protein 1 (ICAT) silence in Wnt signaling pathway and sterol drug NSC67657 induced cell differentiation of HL60 cell.
Methods:HL-60 cells were treated with NSC67657, the cell surface antigen CD14 expression was detected by flow cytometry. Lentivirus LV-ICAT-RNAi vector was constructed and infected HL-60 cells. Then the ICAT gene and protein expression were analyzed using real-time qPCR and Western blot technique. Furthermore, Co-immunoprecipitation assay was used to confirm the interaction of β-catenin/ICAT proteins, and Western blot was employed to compare the expressions of Wnt signaling pathway downstream targets Cyclin D1, TCF-1 and c-Jun between Lentivirus LV-ICAT-RNAi vector infected HL-60 (HL-60i) cells and un-infected HL-60 (HL-60v) cells. The cellular differentiation of HL-60i and HL-60v cells treated with NSC67657 for 24 h was evaluated by Wright’s staining, transmission electron microscopy and flow cytometry analysis.
Results:HL-60 cells could be induced to differentiate into monocytes by 10 μmol/L NSC67657. The CD14 positive cells could reach to (92.30±5.14) % after NSC67657 treatment for 5d. The co- immunoprecipitation assay demonstrated that ICAT protein did interact with β-catenin protein, and the absorbance of protein electrophoresis bands increased in differentiated cells. The expressions of Wnt signaling pathway downstream target proteins in HL-60i cells were higher than that in HL-60v cells when they were treated by 10 μmol/L NSC67657, but lower than NSC67657 untreated cells. CD14 positive HL-60i cells were significantly lower than that of HL-60v cells[ (8.33±3.14) % vs (19.08±4.73) %]when treated with NSC67657, but still higher than that of uninfected and untreated HL60 cells[ (0.60±0.03) %] (F=119.24, P=0.010) . The results of cellular morphology and ultrastructure observation were also in accord with that of cell surface antigen analysis.
Conclusions:ICAT does participate in HL-60 cells monocytic differentiation induced by NSC67657, and Wnt/β-catenin signaling pathway might play a bridge role.
