Influence of YB-1 protein on the biological behaviour in K562/A02 cells.
- Author:
Hui-Ling SHEN
1
;
Lei-Lei ZHOU
;
Qiao-Yun CHEN
;
Chen CHEN
;
Li-Li FANG
;
Xin-Jian FANG
;
Wen-Lin XU
Author Information
1. Department of Hematology, Jiangsu University People Hospital, Zhenjiang 212002, Jiangsu Province, China.
- Publication Type:Journal Article
- MeSH:
ATP-Binding Cassette, Sub-Family B, Member 1;
metabolism;
Apoptosis;
Cell Proliferation;
Genetic Vectors;
Humans;
K562 Cells;
RNA, Small Interfering;
genetics;
Transfection;
Y-Box-Binding Protein 1;
genetics;
metabolism
- From:
Journal of Experimental Hematology
2011;19(2):342-347
- CountryChina
- Language:Chinese
-
Abstract:
The aim of this study was to investigate whether the growth, apoptosis and sensitivity to anticancer agent could be altered after introduction of YB-1 shRNA eukaryotic expression vector into the K562/A02 cells, and its possible molecular mechanisms. The recombinant eukaryotic expression plasmids including YB-1 shRNA and the vector-random-sequence were introduced into K562/A02 cells by lipofectamine mediation, and the positive clones were screened by G418. RT-PCR and Western blot were employed to detect the expression of mRNA and protein of YB-1 in leukemia cells, respectively. The proliferative ability of the cells was determined by MTT assay and cell cycle analysis. Apoptosis of K562/A02 cells was assayed by AnnexinV-FITC/PI double labeled flow cytometry. The drug sensitivity to anticancer agent was determined by MTT assay. The expressions of MDR1 gene and P-gp were detected by RT-PCR and flow cytometry respectively. The results indicated that the levels of mRNA and protein of YB-1 decreased dramatically in three groups of positively transfected cells when compared with control cells. The inhibitory rates of 3 different shRNA sequences targeting YB-1 gene were (65.1 ± 2.1)%, (27.4 ± 1.3)% and (67.4 ± 1.6)% respectively. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased levels of the proliferative ability in K562/A02 cells, and displayed higher at G(1), lower at G(2) and S phase in cell cycle distribution in comparison with the control groups. AnnexinV/PI detection indicated higher AnnexinV(+) ratio in 3 groups of positively transfected cells after being treated with As(2)O(3) of 0.5 µmol/L for 24 hours. The IC(50) values of doxorubicin in 3 groups of positively transfected cells were significantly lower than that in control group. The level of MDR1 gene and P-gp decreased significantly in 3 groups of positively transfected cells. It is concluded that the transfection with YB-1 shRNA gene can inhibit the proliferation of leukemia cells and induce cell apoptosis. The expression of MDR1 mRNA and P-gp decrease after transfection of YB-1 shRNA into K562/A02 cells.
