The Effect Of Per2 On The Phenotype Of K562 Through The Involvement Of C/EBPα And Its Mechanism

In the treatment of chronic myeloid leukemia(CML), blast crisis has been a difficult clinical problem . A new gene targeting CML can be a different treatment strategy. Enhancer binding proteinα(C/EBPα) plays an important role in the proliferation and differentiation of the occurrence of leukemia. In acute myeloid leukemia, C/EBPαregulates the expression of circadian genes Per2, while its regulation was unclear in CML. The low expression of C/EBPαand Per2 as well as their mutual regulation provide a new method in the treatment of CML blast crisis. Therefore, focused on Per2, the downstream targets of C/EBPα, it is of great significance to clarify the effect of Per2 and C/EBPαin CML.Objective:To investigate the effects of the exogenous expression of C/EBPαon the phenotype of leukemia K562 cells and its relationship with related genes, especially the important role of Per2 in leukemia. It will provide experimental evidence in the research of blast crisis of CML for Per2, as a new element of signal transduction.To investigate the function of Per2 on proliferation, apoptosis and differentiation of chronic myeloid leukemia K562 cells, and the effects on the growth of mice K562-induced subcutaneous tumors and leukemia model in nude mice in vivo.To provide a new idea for clarify the pathogenesis and treatment of Per2 in CML.Methods:1 The effects of exogenous expression of C/EBPαon the K562 cells and the change of related genes(1) The C/EBPαexpression plasmid pEGFP- C/EBPαand empty control plasmid were respectively transfected into K562 cells with cationic liposome, and the resistant cells stably expressing the C/EBPαgene were obtained by G418 selection. The morphological changes were observed under light microscope following Wright-Giemsa staining. Flow cytometry was performed to analyze cell cycle distribution and apoptosis and differentiation antigen CD11b. The electron microscopy was used to detect cell apoptosis and coherent genes were respectively detected by RT-PCR and Western blot both at the mRNA and protein level.(2) The specific and non-specific SiRNA sequences were designed, and the plasmids of SiPer2 were constructed. The constructed plasmids were transfected into the pEGFP- C/EBPαK562 cells, pEGFP- C/EBPαK562 cells with red fluorescent light were selected.The changes of genes were detected before and after interfering Per2 mRNA.2 The effect of Per2 gene on the proliferation and apoptosis in the K562 cells and its mechanism of anti-leukemia.The Per2 expression plasmid pcDNA3.1-Per2 and empty control plasmid were respectively transfected into K562 cells with cationic liposome, and the resistant cells stably expressing the Per2 gene were obtained by G418 selection. The morphological changes were observed under light microscope following Wright-Giemsa staining. Trypan blue excluding staining and MTT assay were employed to evaluate cell proliferation. Flow cytometry was performed to analyze cell cycle distribution and cell apoptosis, the electron microscopy was used to detect cell apoptosis. Meanwhile, the expression of the proliferation and apoptosis associated proteins, such as P53, CyclinB1 and C-myc, were respectively detected by RT-PCR and Western blot both at the mRNA and protein level.3 The research about Per2 gene's effect on the tumorigenic ability and tumor growth in the chronic myeloid leukemia mouse modelThree kinds of cells including pcDNA3.1-Per2-K562 cells, pcDNA3.1-K562 cells and the control K562 cells were injected into mice separately through the subcutaneous and tail vein, subcutaneous tumors and leukemic models were formed.The tumorigenicity and tumor growth, physiological changes, as well as bone marrow, liver and spleen and other organs change and the survival time of mice were observed.Results:1 The K562/ C/EBPαcell line stably expressing the Per2 gene was screened out. As compared with either the empty plasmid transfected group(K562/empty)or the untreated group(K562/untreated), the expression of C/EBPαcan promote the differentiation of K562 cells and the expression of differentiation antigen CD11b. Cell cycle analysis showed that the percentage of K562 cells in G2 phase was increased [K562/C/EBPαgroup (17.18±3.5)%, K562/empty group (10.86±2.9)%, untreated group (9.59±2.3 )%, P < 0.05], and distinguished apoptotic peak was seen meanwhile. Flow cytometry showed more apoptosis in the transfected group( 21.1%)than that of K562/empty group( 6.0%)and untreated group(4.2%), P<0.05. Nuclear condensation, fragmentation, karyopycnosis and apoptotic bodies could readily be found under the transmission electron microscope in the K562/C/EBPαgroup. Per2 was significantly elevated by RT-PCR and Western blot both at the mRNA and protein level, while CyclinB1 and C-myc was down-regulated. The plasmid of SiRNAPer2 was successful transfected into stable cell lines K562/C/EBPαcells with more than 30% efficiency.The red fluorescent marker of SiRNA-Per2-K562/C/EBPαcell lines was collected.After specifically interferencing Per2 gene, CyclinB1 and C-myc gene mRNA and their protein expression were significantly raised, while the P53 expression was down- regulated.2 The K562/Per2 cell line stably expressing the Per2 gene was screened out. As compared with either the empty plasmid transfected group (K562/empty) or the untreated group(K562/untreated), K562/Per2 cell was smaller than other groups, cellular growth and proliferation were inhibited ,while no obvious cellular differentiation. Cell cycle analysis that the percentage of K562 cells in G2/M phase was increased [K562/Per2 group (36.04±5.5) %, K562/empty group (12.48±2.7) %, untreated group (9.71±2.1)%, P<0.05]. FCM showed more apoptotic rate in the transfected group[14.8%, P<0.05].Nuclear condensation, fragmentation, karyopycnosis and apoptotic bodies could readily be found under the transmission electron microscope in the K562/Per2 group. P53 was significantly elevated by RT-PCR and Western blot both at the mRNA and protein level, while CyclinB1 and C-myc was down- regulated.3 The mice models of subcutaneous tumors and leukemia were successfully constructed.In mouse model of subcutaneous tumors, the volume and weight of transfected group was significantly lower than the control group and empty vector group, and more apoptosis cells in the transfected group by TUNEL. In mouse model of leukemia, the cellular proliferation of bone marrow was decreased significantly in the transfected group of Per2, so did the liver and spleen infiltration. On the contrary, the survival period of mice was longer than other groups.Conclusion:1 The expression of enhancer binding protein C/EBPαcould not only promote cellular differentiation, but also arrest cell cycle and induce apoptosis in K562 cells .C/EBPαupregulate the Per2 clock gene expression and inhibit CyclinB1, C-myc gene expression. The regulation on the downstream genes of C/EBPαwas affected through the interference of Per2. A possible signal transduction pathway of C/EBPα→Per2→downstream target gene→cell phenotype may exist.2 Exogenous expression of the circadian clock gene Per2 could not only inhibit the growth and proliferation, but also induce massive apoptosis in K562 cells through its regulation on the cell cycle associated genes such as P53,CyclinB1,C-myc etc. and subsequent inhibition on cell cycle progression.3 In animal experiments, Per2 inhibited subcutaneous tumor growth and promote cell apoptosis. In leukemia mice, Per2 inhibited the cell proliferation of bone marrow, the infiltration of liver and spleen and prolonged the survival period of mice. Per2 acts as a tumor suppressor and plays a key role in the leukemia.