[Gfi-1 expression in leukemia patients and inhibitory effects of lentiviral vector mediated silence of Gfi-1 gene on proliferation in K562 cells].
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.
Zhan
Rong
R
Department of Hematoloy, Fujian Medical University Union Hospital, Fuzhou 350001, Fujian Province, China. deanzhanrong@yahoo.com.cn
Wu
Shun-Quan
SQ
Huang
Hao-Bo
HB
Huang
Shen-Lin
SL
Lin
Jun
J
D000293:Adolescent; D000328:Adult; D000368:Aged; D016022:Case-Control Studies; D049109:Cell Proliferation; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D020014:K562 Cells; D016086:Lentivirus / Q000235:genetics; D007938:Leukemia / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D010957:Plasmids; D000091402:Prohibitins; D034741:RNA, Small Interfering / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*; D055815:Young Adult
D004268:DNA-Binding Proteins; C498275:GFI1 protein, human; C000717331:PHB2 protein, human; D000091402:Prohibitins; D034741:RNA, Small Interfering; D014157:Transcription Factors