Aml1b And Aml1-eto Of Pig7 Gene Transcriptional Regulation And Gene Pig7 Leukemia Cell Proliferation Activity Of Research

Objective:To determine the role of AML1b/AML1-ETO protein in pig7 gene transcription,characterize the binding relationship between AML1b/AML1-ETO protein and the pig7 enhancer and clarify the mechanistic roles of pig7 in regulating proliferation of leukemic cells with functional assays.Methods:The expressive plasmids of AML1b and AML1-ETO were transfected into CV-1 and 293 cells.The expression level of endogenous pig7 gene was detected by Realtime-PCR.The luciferase reporter plasmids containing pig7 enhancer/corresponding mutant sequences were constructed and co-transfected into CV-1 cells with expressive plasmids of AML1b and AML1-ETO.The transactivity of pig7 enhancer was assayed by luminometer.Lentiviral vectors pCDH-pig7 containing the open reading frame(ORF) of pig7 and pCDH-anti-pig7 containing antisense RNA sequence of pig7 were constructed to transfer pig7 into leukemic cells and block endogenous pig7 expression respectively,to study its biological functions.Results:Transient expression of exogenous AML1b up-regulated the expression of endogenous pig7 in 293 and CV-1 cells.The specific site required for AML1 protein-DNA binding is located between-1511 and-1503 in pig7 gene.AML1b exhibited a distinct transaetivity to pig7 enhancer with a sequence-specificity and dosage-dependant manner.AMLI-ETO showed no any transaetivity but antagonized the effect of AML1b causing marked reduction of pig7 expression. Utilizing pCDH lentivirus system,we succeeded in generating pCDH-pig7 as well as the RNA interference vector pCDH-anti-pig7.The infection tests showed that ectopic expression of pig7 protein increased apoptosis and promoted differentiation of Kasumi-1 cell in vitro,but no changes in NB4 cell.However, combined use of ATRA and pCDH-pig7 in NB4 cell was also found to cause increased apoptosis and promoted differentiation.Down-regulation of endogenous pig7 by use of pCHD-anti-pig7 increased resistance of Kasumi-1 and NB4 cells to chemotherapy drugs cytotoxicity. Conclusion:AML1b protein up-regulate expression of pig7 gene by binding at specific site between-1511 and-1503 in pig7 gene.AML1-ETO may interfere with the transactivation of AML1b.Pig7 could increase apoptosis and promote differentiation of Kasumi-1 cell witch expressing AML1-ETO and may in turn exert anti-tumor efficacy in a synergical manner with chemotherapeutic agents.