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Published on: June 9, 2021
[Influence of YB-1 protein on the biological behaviour in K562/A02 cells]
Hui-Ling Shen1, Lei-Lei Zhou, Qiao-Yun Chen
1Department of Hematology, Jiangsu University People Hospital, Zhenjiang 212002, Jiangsu Province, China.
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.
Insights
Introducing YB-1 shRNA into K562/A02 leukemia cells inhibits proliferation and induces apoptosis. This approach also reduces multidrug resistance by decreasing MDR1 gene and P-glycoprotein expression, offering potential therapeutic strategies.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Therapy
Background:
- Y-box binding protein 1 (YB-1) is implicated in cancer progression and drug resistance.
- K562/A02 cells are a human chronic myeloid leukemia cell line.
- Understanding YB-1's role is crucial for developing novel leukemia treatments.
Purpose of the Study:
- To investigate the effects of YB-1 shRNA on K562/A02 cell growth, apoptosis, and sensitivity to anticancer agents.
- To elucidate the molecular mechanisms underlying these alterations, including MDR1 gene and P-gp expression.
Main Methods:
- YB-1 shRNA eukaryotic expression vector was introduced into K562/A02 cells.
- Quantitative reverse transcription PCR (RT-PCR) and Western blot analyzed YB-1 expression.
- MTT assay, cell cycle analysis, and AnnexinV-FITC/PI flow cytometry assessed proliferation and apoptosis.
- Drug sensitivity and MDR1/P-gp expression were evaluated.
Main Results:
- YB-1 shRNA significantly reduced YB-1 mRNA and protein levels in transfected cells.
- Cell proliferation decreased, with cell cycle distribution shifting towards G1 phase.
- Apoptosis was induced, particularly after treatment with arsenic trioxide (As(2)O(3)).
- Sensitivity to doxorubicin increased, and MDR1 gene and P-gp expression were significantly downregulated.
Conclusions:
- Transfection with YB-1 shRNA effectively inhibits leukemia cell proliferation and induces apoptosis.
- Downregulation of YB-1 reduces multidrug resistance by decreasing MDR1 mRNA and P-gp expression.
- Targeting YB-1 presents a promising strategy for overcoming drug resistance in leukemia.
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