Molecular Mechanisms Of Aberrant Expression Of Annexin Ⅱ In Acute Promyelocytic Leukemia

Objective: Acute promyelocytic leukemia （APL） is characterized by PML/RARαfusion gene formed by t（15:17） translocation and life-threatening bleeding diathesis.The severe coagulopathy is due to predominant hyperfibrinolysis and mild disseminatedinvascular coagulation, attributed mostly to aberrant expressions of annexin II andtissue factor in APL cells. Previous research has discovered that tissue factor is elevatedby the specific PML/RAR ɑfusion protein, however the association of annexin IIexpression and the oncoprotein remains unknown. The current project was toinvestigate the association between annexin II and PML/RAR ɑfusion protein, byexaming the involvement of PML/RARɑ fusion protein in annexin II upregulation.Methods:1. To testify the association between annexin II and the oncoprotein,U937-PR9cells were incubated with100μM ZnSO4, an inducing agent of PML/RARα,for0h,0.5h,1h,2h,6h,12h and24h, and the expressions of PML/RARα and annexin IIwere evaluated at mRNA and protein levels, using QT-PCR and western blot,respectively, to determine the transactivation of annexin II by PML/RARα fusionprotein.2. A group of truncated promoters of annexin II were inserted into pGL3-basicplasmid, a luciferase gene vector, according to stepwise promoter deletion principle.The constructs include the promoter truncations as follows:-1074bp⁺46bp,-641bp⁺46bp,-374bp⁺46bp,-198bp⁺46bp,-91bp⁺46bp. The correctness ofinserts was verified with restriction endonuleases of KpnI and XhoI, and subsequentDNA sequencing.3. Constructs containing annexin II promoter truncations weretransiently co-transfected with pSG5-PML/RARα and pRL-SV40, an internal control, into COS-7cells. The transactivation of annexin II promoter by the oncoprotein wasthen analyzed with dual-luciferase activity assay.Results: The induction of PML/RARα expression was detected1hour after100μM Zn²⁺incubation, followed by Annexin II expression at2hours, indicating thatannexin II upregulation is the downstream event of the oncoprotein expression. Thedual-luciferase activity assay showed that annexin II promoter was transactivated by theoncoprotein.Conclusion: In U937-PR9cells, expression of Annexin II is up-regulated byPML/RARα fusion protein through transactivating on its promoter region.