Gfi-1 expression in leukemia patients and inhibitory effects of lentiviral vector mediated silence of Gfi-1 gene on proliferation in K562 cells.
- Author:
Rong ZHAN
1
;
Shun-Quan WU
;
Hao-Bo HUANG
;
Shen-Lin HUANG
;
Jun LIN
Author Information
1. Department of Hematoloy, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian Province, China. deanzhanrong@yahoo.com.cn
- Publication Type:Journal Article
- MeSH:
Adolescent;
Adult;
Aged;
Case-Control Studies;
Cell Proliferation;
DNA-Binding Proteins;
genetics;
Female;
Gene Silencing;
Genetic Vectors;
Humans;
K562 Cells;
Lentivirus;
genetics;
Leukemia;
genetics;
pathology;
Male;
Middle Aged;
Plasmids;
RNA, Small Interfering;
genetics;
Transcription Factors;
genetics;
Young Adult
- From:
Journal of Experimental Hematology
2010;18(4):849-854
- CountryChina
- Language:Chinese
-
Abstract:
This study was aimed to quantitatively detect the expression levels of gfi-1 gene in leukemia patients, and to investigate the effect of gfi-1 gene silenced by short hairpin RNA (shRNA) on proliferation of leukemia cell line K562. Quantitative real-time PCR and Western blot were used to detect the mRNA and protein expression levels of GFI-1 in newly diagnosed patients with leukemia. One pair of oligonucleotide sequences targeted at human gfi-1 mRNA were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pLVTHM vector. Virus particles were collected when the control and shRNA vectors had been co-transfected with the psPAX2 packaging plasmid and the envelope plasmid pMD2 G into HEK-293T cells using Lipofectamine 2000. The K562 cells were transfused with 1 x 10⁶ recombinant lentivirus-transfusing units plus 6 microg/ml of polybrene. Rea-time PCR and Western blot were used to detect the expressions of gfi-1 and bax mRNA after lentivirus transfusion. CCK-8 assays was used to evaluate the proliferation potential of cells. The results showed that the gfi-1 expression level in all leukemia patients was significantly higher than that in normal group (p < 0.05); the gfi-1 mRNA expression in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL) patients was significantly higher than that in normal group (p < 0.05); but the difference of gfi-1 mRNA expression between AL and CML or ALL and AML was not significant. Notably, the gfi-1 mRNA expression level had a positive correlation with high white blood cell count of > 20.0 x 10⁹/L (p < 0.05). As was expected, the mRNA and protein level of gfi-1 was reduced significantly in K562 cells after lentivirus transfusion, whereas the mRNA and protein level of bax was upregulated. And CCK-8 assay showed that gfi-1 gene silencing can inhibit K562 proliferation. It is concluded that gfi-1 expression is upregulated in leukemia patients and may contribute to leukemogenesis. The gfi-1 specific shRNA mediated by lentivirus can effectively down-regulate the expression of gfi-1 and inhibit the proliferation of K562 cells, which lay a basis for further research on gene therapy in leukemia.
