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, 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 our protophase study, we found that Twist1 should be a positive regulator in the development of hematopoietic malignancies. In this study, we detected Twist1 expression in various leukemia patients specimens, to explore the correlation between Twist1 and leukemia cell differentiation.Twist1 gene expression was detected by real-time quantitive RT-PCR (RQ-RT-PCR) in bone marrow cells of 226 patients including 102 AML patients,56 CML patients,29 ALL patients,6 MDS patients,3 healthy donors and 29 ITP patients were used as normal control. The relationship between Twist1 expression and the established prognostic factors such as age, karyotype, CD34 expression, blast percentage and FAB subtypes was explored.We found that either disease status, or karyotype were correlated with the expression of Twist1.With respect to FAB cytotypes, expression of Twist1 gene in CML, AML was statistically higher than that in normal and other subtypes (p<0.05).The expression of Twist1 in M2, M3 and M5 patients was much higher than that of normal controls, (p< 0.05). Moreover, a significant difference in Twistl gene expression was obtained between the AML-ETO positive group and the AML-ETO negative group 0.0390(0.0270-0.7060) verse 0.8000(0.0060-29.34), (p<0.05). The Twistl expression of BCR/ABL positive patients was significantly higher than that BCR/ABL negative patients 0.5330 (0.0290-9.349) verse 0.1205 (0.0130-21.27), (p<0.05).The Twist1 expression of PML/RARa positive patients 0.5785 (0.0360-13.16) was significantly lower than that of PML/RARa negative 1.0070 (0.0370-31.14), but there was no statistic differences (p>0.05).The Twistl expression of CD34 positive patients was higher than that of CD34 negative 0.3765 (0.0060-45.88) verse 0.1400 (0.0220-30.74), but there was no statistic differences (p>0.05).Twistl expression was independent of the blast percentage in diagnostic bone marrow (p>0.05).Twistl gene expression was detected by real-time quantitive RT-PCR (RQ-RT-PCR) and immunohistochemistry in bone marrow cells of 33 patients including 11 AML patients,8 CML patients,7 ALL patients, and 7 ITP patients were used as normal control. The result shows that the expression of Twistl mRNA was highly related to the expression of Twistl protein, that means the expression of Twistl mRNA could reflect the expression level of Twistl protein (y=0.01959 x-0.1580, p=0.0211).Conclusion, high expression of Twistl in CML, M2, M3 and M5 patients provides novel insights in the diagnosis of leukemia and leukemogemesis study.We investigated the relationship between the expression of Twistl and differentiation of leukemia cells using TPA which had apparent effect in megakaryocyte differentiation in K562 cells. Then realtime PCR was used to detect the expression of Twistl, mda-7/IL-24. Significant increased expression of mda-7/IL-24, and decreased expression of Twistl was observed in K562 cells which treated with TPA, compared with non-TPA control cells. The expression of CD41, CD61 detected by FACS, were apparently higher than non-TPA control, suggest that K562 cells were induced to undergo megakaryocytic differentiation. K562 cells were transfected with mda-7/IL-24-targeted siRNA or negative control siRNA by using lipofectamine transfection, at the same time TPA was added into each system to induce the expression of mda-7/IL-24. mda-7/IL-24-targeted siRNA specifically reduced IL-24 expression by 80% as compared with control groups. The expression of Twistl decreased, indicates that mda-7/IL-24 could inhibit the expression of Twist1.These data suggest thatIn order to prove phenomena above, we constructed pcDNA3.1-myc-his (-)B-Twistl plasmid, and transfect it into K562 cell line, to study the relationship of Twist1, mda-7/IL-24 and cell differentiation.Taken together, these data strongly support that Twistl should be implicated in the differentiation of luekemia cells. Twistl might be part of the reason that mda-7/IL-24 could induce differentiation of luekemia cells.