The Regulation Effects Of WISp39 Gene In Human Leukemia Cells

PartⅠConstruction of a Eukaryotic Expression Vector pLenti6/V5-WISp39 and Its Transfection into U937 Cell LineObjective: To study the effect of WISp39 gene on proliferation of the leukemia cells, a eukaryotic expression vector pLenti6/V5-WISp39 was constructed and transfected into the human myelocytic leukemia cell line-U937 cells.Methods: The plasmid pLenti6/V5-WISp39 was constructed by gene-recombination technology and verified by sequencing. pLenti6/V5-WISp39 and pLenti6/V5-LacZ (control) were transfected into the U937 with Lipofectamine2000? while another untransfected group as the blank. After transfecting, the cells were cultured in 37℃with 5% CO2. Cell suspension was changed 6 hours later and the cells were collected 48 hours after transfection. The change of WISp39 mRNA level in different groups was evaluated by Real-time PCR.Results: The sequence of the recombinant was proved to be correct according to PCR and the sequence analysis. WISp39 mRNA level in experimental group was increased by 5.5±1.2 folds.Conclusion: The eukaryotic expression vector pLenti6/V5-WISp39 was successfully constructed and transfected into the human myelocytic leukemia cell line-U937 cells, which provide an approach for the research of WISp39 gene. PartⅡEffects of WISp39 on Proliferation, Cell Circle and Apoptosis in U937 CellsObjective:To study the role of WISp39 in the regulation of proliferation, apoptosis and cell cycle in the leukemia cell line -U937 cells.Methods: 48 hours after transfecting the plasmid pLenti6/V5-WISp39 into the U937 cells, we collected the cells and tested changes of the cells in the three different groups. Proliferation of the cells was analyzed by CCK-8. Apoptosis of the cells, marked with Annexin V and PI, was detected by flow cytometry. And cell cycle, with the cells marked with PI staining solution, was also detected by flow cytometric analysis.Results: Plasmid pLenti6/V5-WISp39 could readily enhance the expression of WISp39 in U937 cells. A significant growth inhibition (37.6%) was observed in cells tranfected with pLenti6/V5-WISp39, while in the control group, plasmid pLenti6/V5-lacZ showed little effect on U937 growth. Further study revealed the fact that pLenti6/V5-WISp39 had no significant apoptosis induction effect on U937 (P>0.05), however, it could regulate U937 proliferation via cell cycle modulation. Compared with pLenti6/V5-lacZ, pLenti6/V5-WISp39 increased G0/G1 fraction by 10% 48 hours after transfection (40.59±0.7% versus 49.79±1.1%, P<0.05).Conclusion: WISp39 gene could suppress the U937 proliferation and arrest cell cycle at G0/G1 phase, while it didn't change the apoptosis rate, which meant WISp39 could act as a negative regulate factor in tumour cells.