Effect Of MLL-AF9 Fusion Gene On The Proliferation Of Acute Monocytic Leukemia Cell And Initial Study On The Molecular Mechanism

PART I Identification on the Sequence of MLL-AF9 Fusion Gene in Acute Monocytic Leukemia Cell Line THP-1Objective: Clone the MLL-AF9 fusion gene from the acute monocytic leukemia cell line THP-1 and analyze its junction sequence.Methods: RT-PCR was used to separate MLL-AF9 fusion gene from the acute monocytic leukemia cell line THP-1 and clone it into T vector for sequence analysis.Results: MLL-AF9 fusion gene has been cloned from the acute monocytic leukemia cell line THP-1. Sequencing showed that the fusion gene was a direct in-frame fusion between exon 9 of MLL and exon 5 of AF9.Conclusions: Clone and sequence the MLL-AF9 fusion gene from THP-1 successfully. It is necessary to design the siRNA targeted MLL-AF9 in the next part. PART II Effect of Down-regulating MLL-AF9 Fusion Gene on the Proliferation of Acute Monocytic Leukemia Cell Line THP-1Objective: To study the effect of MLL-AF9 fusion gene on the proliferation of human acute monocytic leukemia cell line THP-1.Methods: One group of siRNA was designed targeting MLL-AF9 mRNA and finally obtained by chemosynthesis. Then the obtained siRNA was transfected into cultured THP-1 by lipofectamine. Flow cytometry was used to detect siRNA transfection efficiency. The level of MLL-AF9 mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). Cell proliferation rate was analyzed by MTT assay. Hoechst33258 staining was used to observe the apoptotic bodies in the cells. The change of apoptosis rate and cell cycles was detected by flow cytometry.Results: SiRNA transfection efficiency was 69.1%±1.8%. The level of MLL-AF9 mRNA expression was significantly inhibited in siRNA-treated cells as compared with that in the controls (P<0.01). The percentage of G0/G1 phase cells was significantly increased in siRNA-treated cells in comparion with that in the control cells (P<0.01), but the percentage of S phase cells was significantly decreased (P<0.01). MLL-AF9-targeted siRNA inhibited the proliferation of THP-1 cells and induced cell apoptosis effectively after transfection. Staining of the siRNA-treated cells with Hoechst 33258 showed the morphology characteristic to apoptosis.Conclusions: MLL-AF9 fusion gene sustains and promotes the proliferation of human acute monocytic leukemia cell line THP-1. PART III MLL-AF9 Fusion Gene Regulates Cyclin-Dependent Kinase Inhibitor p27 Expression in Acute Monocytic Leukemia Cell Line THP-1Objective: To explore whether MLL-AF9 oncegene regulates p27 expression in THP-1 cell line.Methods: SiRNA targeting MLL-AF9 were designed and constructed, and transfected into THP-1 by lipofectmine. Flow cytometry was used to detect siRNA transfection efficiency. The level of MLL-AF9 mRNA expression was examined by reverse transcription polymerase chain reaction (RT-PCR) and the expression of MLL-AF9 and p27 protein was detected by Western blot. Chromatin immunoprecipitation (ChIP) assays was used to confirm whether MLL-AF9 binds to the p27 promoter in THP-1 cell line.Results: The level of p27 expression shows up-regulation at both the mRNA level and protein level after MLL-AF9 expression was significantly inhibited in siRNA-transfected cells as compared with that in the controls (P<0.01). MLL-AF9 binds to the p27 promoter in THP-1 cell line.Conclusion: MLL-AF9 oncoprotein in THP-1 cells inhibits the p27 expression, and regulates p27 promoter activity.