[Effect of YB-1 gene knockdown on human leukemia cell line K562/A02].
OBJECTIVE
To investigate the potential effects of YB-1 gene knockdown on gene expression profile, cell growth and apoptosis in leukemia cell line K562/A02.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Xu
Wen-lin
WL
Department of Hematology, the Affiliated People's Hospital, Jiangsu University, Zhenjiang, Jiangsu, 212002 P.R. China.
Zhou
Lei-lei
LL
Chen
Qiao-yun
QY
Chen
Chen
C
Fang
Li-li
LL
Fang
Xin-jian
XJ
Shen
Hui-ling
HL
D017209:Apoptosis; D002453:Cell Cycle; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D055785:Gene Knockdown Techniques; D006801:Humans; D007938:Leukemia / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D051840:Y-Box-Binding Protein 1
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D051840:Y-Box-Binding Protein 1; C070856:YBX1 protein, human