[The down regulation of VEGF gene expression may link to change the expression profile of genes in leukemia cell line K562].
OBJECTIVE
To explore the potential effects of anti-VEGF hairpin ribozyme gene to gene expression profiles in leukemia cell line K562.


METHODS
The lipofectamine mediation was used to transfect the recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the non-recombinant vector as control into K562 cells. And the positive clones were screened by G418. Ribozyme gene in K562 cells was confirmed by PCR. Fluorescent real time reverse transcription-PCR(RT-PCR) and Western blotting were employed to detect the expression of VEGF mRNA and protein in leukemia cells. cDNA microarray was used to explore the alteration of gene expression profiles when decreasing VEGF gene expression in leukemia cells. Expression of PCNA and GSN genes were verified by semi-quantitative RT-PCR.


RESULTS
The pcDNA3-RZ and pcDNA3 had been transfected into the human leukemia cell line K562 and positive clones been screened by G418. Stable expression of the ribozyme gene in K562 cells was confirmed by PCR. The level of VEGF mRNA and protein decreased dramatically in K562-RZ cells when compared with K562 or K562-PC (K562 cell transfected with empty vector) cells. The gene expression profiles were changed by transfection of anti-VEGF hairpin ribozyme gene into K562 cells. Among 4096 gene clones on the microarray, 191 (4.86%) genes were detected to have the marked changes with 104 down-regulated and 87 up-regulated, that were functionally related to cell cycle progression, gene replication, metabolism, cell apoptosis, cell signal transduction, and oncogenes etc. An increased expression of GSN gene and a decreased expression of PCNA gene in K562/RZ cells have been detected by RT-PCR.


CONCLUSIONS
Down-regulation of VEGF gene by introducing anti-VEGF hairpin ribozyme gene can alter the gene expression profiles in K562 cells, leading to change of cell growth, differentiation and apoptosis in K562/RZ cells.
Xu
Wen-lin
WL
Department of Hematology, The Affiliated People's Hospital of Jiangsu University, Zhenjiang, Jiangsu 212002, P.R.China. XWL0806@163.com
Shen
Hui-ling
HL
Wu
Zhao-yang
ZY
Tang
Hua-rong
HR
Wang
Fa-chun
FC
D015536:Down-Regulation / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects*; D020869:Gene Expression Profiling; D006801:Humans; D020014:K562 Cells; D007938:Leukemia / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D016337:RNA, Catalytic / Q000494:pharmacology*; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D016337:RNA, Catalytic; D042461:Vascular Endothelial Growth Factor A; C418481:hairpin ribozyme