Mechanism research and effects of clock gene Per2 in K562 leukemia cells on their proliferation, differentiation and apoptosis
Objective To investigate the effects of circadian clock gene Period2 ( Per2) on the proliferation, differentiation and apoptosis of K562 cells and its probable molecular mechanism. Methods 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 Per2 gene were obtained by G418 selection. Their 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, and electron microscopy was used to detect cell apoptosis. Meanwhile, the expressions of proliferation and apoptosis associated proteins, such as P53, Cyclin B1 and C-Myc, were respectively detected by RT-PCR and Western blot analysis at mRNA and protein level. Results The K562/Per2 cell line stably expressing Per2 gene was screened out. As compared with either the empty plasmid transfected group ( K562/empty) or the untreated group ( K562/untreated), K562/Per2 cells was smaller in volume and showed no obvious cellular differentiation. Circadian clock gene Per2 could significantly inhibit both growth and proliferation of K562 cells. The percentage of K562 cells in G2/M phase increased [ K562/Per2 group (36.1 + 5.5 ) %, K562/empty group (12.5 ± 2.9 )%, untreated group (9.7 ± 2.3)%, P <0. 05] and more apoptotic cells were found in the transfected group ( 14.8%, P <0.05). Nuclear condensation, fragmentation, karyopycnosis and apoptotic bodies were found under transmission electron microscope in the K562/Per2 group. P53 was significantly elevated at the mRNA and protein level, while Cyclin B1 and C-Myc were downregulated. Conclusion Expression of circadian clock gene Per2 can not only inhibit the growth and proliferation of K562 cells, but also induce massive apoptosis possibly through its regulation on cell cycle associated genes and subsequent inhibition on cell cycle progression.
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万方数据
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