Influence of YB-1 protein on the biological behaviour in K562/A02 cells / 中国实验血液学杂志

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.

Mechanisms of preventive effect of tetrandrine on acquired multidrug resistance in K562 cells / 中国实验血液学杂志

This study was purposed to explore the mechanisms of preventive effect of tetrandrine (TTD) on doxorubicin (ADM)-induced multidrug resistance (MDR) in human leukemia cell line K562 from two aspects of the transcription control of MDR1 gene and cell apoptosis. The experiment was divided into 3 groups: group I-blank control; group II-ADM-induced drug-resistance; group III-ADM-induced drug-resistance after pretreatment with TTD. Reverse transcription-PCR (RT-PCR) was used to detect the mRNA expression levels of c-Jun, YB-1 and Survivin genes. Western blot was used to determine the nuclear protein expression levels of c-Jun and YB-1. Flow cytometry was used to assay the apoptosis of cells. The results showed that as compared with group I, the expression levels of c-Jun mRNA and nuclear protein decreased (p < 0.05), as well as the expression levels of YB-1 mRNA and nuclear protein increased in group II (p < 0.05). However, the expression of Survivin mRNA had no change (p > 0.05); the apoptosis rate of cells was 8.31%. As compared with group II, the expression levels of c-Jun mRNA and nuclear protein increased (p < 0.05), expression levels of YB-1 mRNA and nuclear protein as well as Survivin mRNA decreased in group III (p < 0.05). The apoptosis of cells was 97.2%. It is concluded that TTD can inhibit the expression of YB-1 and up-regulate the expression of c-Jun, thus inhibit the expression of MDR1 gene. TTD can also inhibit the expression of Survivin and increase the apoptosis of cells induced by ADM.

Effect of YB-1 gene knockdown on human leukemia cell line K562/A02 / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the potential effects of YB-1 gene knockdown on gene expression profile, cell growth and apoptosis in leukemia cell line K562/A02.</p><p><b>METHODS</b>The recombinant eukaryotic expression plasmid containing YB-1 short hairpin RNA (shRNA) or random-sequence (HK) were transfected into K562/A02 cells by lipofectamine mediation. cDNA microarray was performed to explore the alteration of gene expression profile when YB-1 gene expression was decreased. Expression of CARD8 and RHOC genes were verified by semi-quantitative reverse transcription-PCR (RT-PCR). The proliferative ability of the cells was determined by methyl thiazolyltetrazolium (MTT) assay and cell cycle analysis. Cell apoptosis was assayed by Annexin V-FITC/PI double labeled flow cytometry.</p><p><b>RESULTS</b>The levels of YB-1 mRNA and protein decreased dramatically in three positively transfected cells when compared with untransfected K562/A02 cells or K562/A02-HK thansfected cells. Gene expression profile was altered by transfection of YB-1 shRNA into K562/A02 cells. Among 47,000 genes on the microarray, 252 genes were detected to have changes, with 143 down-regulated and 109 up-regulated. They were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, etc. An increase in CARD8 gene expression and a decrease in RHOC gene expression have been confirmed by RT-PCR in K562/A02-YBX13 cells. The introduction of exogenous YB-1 shRNA gene into K562/A02 cells resulted in decreased proliferation, higher G1, lower G2 and S ratio in cell cycle distribution in comparison with the control groups. Annexin V/PI detection indicated higher Annexin V+ ratio in the three positively transfected cells 24 hours after cells were treated with 0.5 micromol/L of As2O3.</p><p><b>CONCLUSION</b>Down-regulation of YB-1 gene by shRNA-YB-1 can alter the gene expression profile in K562/A02 cells, leading to change of cell proliferation and apoptosis.</p>