Effects Of Notch Gene Overexpression On Proliferation And Mechanism Research In K562 Cells

BackgroundMore and more study have proved that Notch signaling is very important for the stability of hematopoietic system,as sustain the undifferentiated state of Hematopoietic stem cell and decreased as HSCs differentiate. The activation of Notch signaling may have different fuction in different cell type. Notch2 receptor is an important member of Notch signaling family and play a key role in regulating the development process in embryo.Abnormal Notch2 expression is very important for the development of hematopoietic tumor, The expression of Notch2 is necessary for marginal zone B cell development and in related with CD23 overexpression in chronic B cell lymphoid leukemia.Overexpression of ICN2 can induce T cell lymphoid leukemia,and cells are prone to CD8 cells. Notch2 maybe a new tumor suppressor because it is nessary for the antitoxinum and antibiotics.But whether the abnormal expression of Notch2 is involved in K562 cells is not clear.Objective Our study use the K562 cells to study the Chronic myelocytic leukemia1.To investigate the Notch2 expression of human chronic myeloid leukemia cell line K562 cells after transfected with exogenous Notch2;2.To study the effects of overexpression of exogenous Notch2 on cell proliferation;3.To study the effects on cell cycle after transfected with exogenous Notch2 in K562;4.To investigate the possible mechanisms of Notch2 overexpression inhibited the proliferation of K562.MethodsWe grouping the cells in ICN2 transfected group and un-transfected group,and cultured for 24h,48h respectively.1. Exogenous intracellular fragment of Notch2 were transfected in K562 cells with LipofectamineTM2000;2. RT-PCR and Western-blot were used to detect the expression of Notch2 mRNA and protein respectively;3. RT-PCR were used to detect the expression of Hes1,Hey1 target genes and numb,Bcl-2,NF-κB,TGF-β1 genes of K562 cells after transfection;4. Inverted phase contrast microscope was used to observe the morphology change of K562 cells in 24,48h; MTT assays were used to detect the proliferation of K562 cells; 5. Flow cytometry was used to detect the cell cycle of K562 cells after transfected with ICN2 48h.Results1.The 387bp bands appeared clearly after PCR was used to detect the amplifed plasmid;2. The expression of Notch2 mRNA and protein were upregulated in ICN2 transfected group;3. The target gene Hes1,Hey1 were upregulated in ICN2 transfected group;4. As compared with untransfected group,the cell number of transfected K562 cells decreased with the diameter and volume were shrink;the proliferation of K562 cells was inhibited significantly(P<0.001);5. G1 phase cells (P<0.005)increased and S phase cells(P<0.001) decreased after transfected 48h;6.The expression of NF-κB and TGF-β1 mRNA were upregulated,Bcl-2 was downregulated and numb was not changed in transfected group;ConclusionThrough transfected ICN2 in K562 cells with LipofectamineTM2000, we found the expression of Notch2 mRNA and protein are enhanced,and the target gene Hes1,Hey1 are both enhanced in ICN2 transfected group,indicate that Notch2 is expressed in K562 cells successfully.Futher study found that overexpression of Notch2 can slow down the proliferation of K562 cells,and the diameter and volumn were also shrink,cells were inhibited in G1 phase, and we found the expression of TGF-β1,NF-κB were upregulated and Bcl-2 downregulated. Our study may indicated that the exogenous intracellular fragment of Notch2 inhibited the proliferation of K562 cells, affected the cell morphology and inhibit the in G1 phase, possiblely by upregulate the expression of NF-κB,TGF-β1 mRNA and downregulate the expression of Bcl-2.