Expression And Effect Of Twist1 Gene On Hematopoietic Malignances

Basic helix-loop-helix(bHLH) transcription factors have been shown to play an important role in controlling cell type determination and differentiation.The Twist-1 protein(also called Twist) is a highly conserved transcription factor that belongs to the family of basic helix-loop-helix(bHLH) proteins.Since its initial found in Drosophila, the Twist gene has been studied in numerous other solid tumor types,including carcinoma of the lung,breast,stomach,pancreas,prostate and breast,consistently demonstrating growth-promote,promoting migration,anti-apoptosis,EMT effects on various tumor types.Although Twist has been a known candidate cancer gene therapeutic molecule,the effects on leukemia remain elusive.In this study,we detected the expression of Twist1 gene in various leukemia patients.To analyze the expression of Twist1 gene in various leukemia patients and its clinical significance,explore the possible role of Twist in the development of leukemia.A Real-time quantitive RT-PCR(RQ-RT-PCR) method was applied to assay the Twist gene expression levels in bone marrow cells of 147 leukemia patients including 70 AML,30 CML,15 ALL,6 MDS,3 healthy donors and 13 ITP patients were used as normal controls.The relationship between expression levels and the established prognostic factors such as age,tissue infiltration,karyotype,CD34 expression,blast percentage and FAB subtypes was investigated.Either disease status,or age were found to significantly correlate with the expression of Twist.With respect to FAB cytotypes,expression of Twist1 gene in CML,M2,M5(n = 71) was statistically higher than that in normal and other subtypes(n = 76,P＜0.001), median Twist1 expression level of MDS patients was much higher than that of normal controls,but there was no statistic differences.The expression of Twist1 in ALL patients was lower than that in normal controls.(0.1155(0.002-1.521 )verse 0.265(0.018-1.723 )). Moreover,a significant difference in Twist1 gene expression was obtained between the AML-ETO positive group and the AML-ETO negative group(0.121(0.021-0.592)versus 0.596(0.03-23.804),P＜0.05).The Twist l expression of BCR/ABL positive patients was significantly higher than that of BCR/ABL negative(0.608(0.011-56.73) verse 0.298(0.039-20.757)),but there was no statistic differences(p＞0.05).Twist1 expression was independent of the blast percentage in diagnostic bone marrow or peripheral blood white blood cells(WBC)(correlation coefficient R~2 =002).Conclusion Twist1 gene takes a increase in M2,M3,M5 and CML patients with different levels,which provide novel insights in the diagnosis of leukemia and leukemogemesis study.These observations suggest that Twist1 gene over-expression might reflect the convergence of several important unfavorable prognostic factors in leukemia.We investigated the anti-tumor effects of knock down Twist1 gene using shRNA which is constructed into a eukaryotic interfere plasmid,then we using lipofectamine transfection on human leukemic cells in vitro.Significant decrease of tumor cell viability was observed in K562 cells and KGla cells transient transfected with Twist1 shRNA, compared with control cells transfected with psh-H1-copGFP,psh-H1-copGFP-controla,psh-H1-copGFP-controlb or nontransfected cells. Transfection with Twist1 shRNA also inhibits colony formation in vitro in K562 and KGla cell lines.Seventy-two hrs after Twist1 shRNA transfection,a significant increase in Annexin v-positive cells was observed in the K562 lines(11.3±0.05%),compared with control cells(5.6±0.14%),and there was a significant decrease in cell migration percent.However,these 2 cell lines failed to demonstrate any significant change in the cell cycle analysis.Our results show that Twist1 shRNA induces growth suppression and apoptosis in K562 cells.Moreover,the in vivo study showed that K562-Twist1-shRNA had lower oncogenicity in tumor size and weight than K562-V or K562-controla,b(all P＜0.05) in nude mice model.Taken together,these data strongly support that Twist1 should be a positive regulator in the development of hematopoietic malignancies.