The Study Of Function And Molecular Mechanisms Of Annexin Ⅱ Overexprssion In Acute Promyelocytic Leukemia

Objective: Environmental toxicology has changed its focus from simpledose-response relationship to the mechanistic study at molecular level, deriving aintergrative discipline combining basic medicine with clinical medicine. The newworking model of the environmental toxicology is to identify problems through clinicalmedicine, analyze with epidemiological statistics, and solve the problems withadvanced biotechnology. Leukemia, one of the most serious diseases threatening humanhealth, is strongly connected with environmental factors, but the area of research stillneeds to be greatly improved. We used acute promyelocytic leukemia （APL） as adisease model, and through studying the molecular mechanism underlying its bleedingcomplication, we hope to establish a research platform exploring what/howenvironmental factors act in the occurrence of leukemia.Method: Coagulation, is one of the primary causes of early death in APL.AnnexinⅡ is an important factor in the regulation of plasminogen system. Our previousstudy has demonstrated that the PML/RAR ɑfusion gene, a characteristic of APL, couldup-regulate the expression of AnnexinⅡ protein.In the current project, we advancedour research by exploring the functional significance of Annexin Ⅱoverexpression inAPL and underlyhing mechanisms using methods of molecular biology and cellularbiology, as well as establish a systematic platform of experimental methods applicableto the environmental toxicology research. There are two parts in this research: thepathophysiological effects of Annexin Ⅱprotein and the underlying molecularmechanism of Annexin Ⅱ overexpression. The pathophysiological effects of AnnexinⅡprotein were evaluated using plasmin generation assay and invasion assay. Briefly,U937-PR9, when induced with ZnSO₄, expressed PML/RARα fusion protein in vitro, which subsequently up-regulated AnnexinⅡ protein;A stable cell line, overexpressingAnnexinⅡ, was established by electroporating Annexin ⅡcDNA into U937, and thechange in plasmin generation and the rate of cell invasion were analyzed in thecondition of Annexin Ⅱ overexpression. The mechanistic study of AnnexinⅡoverexpression included:1) Construction of recombinant plasmids in dual-luciferasereporter gene with Annexin Ⅱ promoters. The stepwise truncations of AnnexinⅡpromoter region were constructed into the upstream of pGL3-Basic Vector which wascircle plasmid with dual-luciferase reporter genes. Then the products were determinedby degestion with double restriction enzymes and sequencing;2) Transientcotransfection and double luciferase detection. The segments of AnnexinⅡ promotersin dual-luciferase reporter genes, eukaryotic vector plasmid of PML/RARα fusionprotein and internal reference plasmid SV40were co-transfected into COS-7bylipofection. Then transcriptional regulation of AnnexinⅡ promoters by PML/RARαfusion gene were examined as indicated by fluorescence intensity, and thecorresponding base pairs of Annexin Ⅱpromoter region was then determined,establishing a working model for the future study on the regulation of AnnexinⅡpromoter region by PML/RARα fusion gene.Results:1) U937-PR9expressed PML/RARα fusion protein when induced by100μmol ZnSO₄for24hours, accompanied by enhanced plasminogen cleavage and therate of cell invasion;2) The successful construction of recombinant eukaryoticexpression vector and plasmids with dual-luciferase reporter genes. The recombinantplasmids of pcDNA3.1-AnnexinⅡ and pGL3-Annexin Ⅱ promoters （-158bp⁺120bp,-124bp⁺120bp,-62bp⁺120bp,-34bp⁺120bp,-15bp⁺120bp and+3bp⁺120bp）were confirmed by double restriction enzyme digestion and sequencing;3) Theexpression of AnnexinⅡ in the stable cell line were confirmed by RT-PCR and WesternBlot, as reflected by increased expression of Annexin Ⅱgene and protein in U937（ANX2） compared with normal U937;4) Protein functional experiments. The plasmingeneration assay showed that AnnexinⅡ could improve plasminogen cleavage with theinteraction of t-PA, while antibodies against Annexin Ⅱcould significantly reduce theactivity. This implys that AnnexinⅡ may underlie hyperfibrinolysis in APL. The cellinvasion assay indicated that U937overexpressing Annexin Ⅱacquired highermigration rate and mobility, while antibodies against Annexin Ⅱcould abolish theactivity, implicating that AnnexinⅡ may involve in infiltration and metastasis of tumorcells;5) Dual-luciferase reporter gene activity assay revealed that PML/RARα fusion gene could significantly up-regulate AnnexinⅡ promoters, especially from the124ndbase upstream of transcriptional start site, indicating the regulatory region of AnnexinⅡpromoters by PML/RARα fusion gene may reside between the34thand124thbaseupstream of transcriptional start site. However, the sequence of the DRE and regulatoryworking model are yet to be determined.Conclusion: Overexpression of Annexin Ⅱprotein could improve plasmingeneration and induce primary hyperfibrinolysis, which may be the major reason of thebleeding in APL. Annexin Ⅱpromoted the invasion of tumor cells, implying thepossible involvement of AnnexinⅡ in infiltration and metastasis of leukemia.PML/RARα fusion gene up-regulated AnnexinⅡ expression at transcriptional levelthrough interacting with AnnexinⅡ promoter. Through evaluating the functionalsignificance of AnnexinⅡ and identifying the mechanism of Annexin Ⅱ up-regulationin APL, we established a working research platform applicable to the study ofmolecular mechanisms underlying leukemia which may be environmentalfactors-related, and therefore set solid foundation for the future mechanistic study ofenvironmental pollutants in the etiology of leukemia.