Effect Of SiRNA-mediated Silencing Of Bmi-1 Expression On The Autophagy Of K562 Cells

Objective: Autophagy, as the degradation of intracellular components by lysosomes, plays some distinct processes role in cells. In general, autophagy play a certain role in the self-renew of normal cells, but when the environment changes, autophagy without control in cells can cause cell metabolism circulation suffocate and cell death. The B lymphoma Mo-MLV insertion region 1 homolog(Bmi-1) as a member of the Polycomb Group family was originally known as an oncogene, recent study have shown that Bmi-1 played a critical role in stem cell proliferation, differentiation, aging and in a variety of the development of tumor. In our study, Chronic myelogenous leukemia(CML) K562 cell line was used to study Bmi-1 gene is involved in the autophagy of K562 cells and further to explore its possible molecular mechanisms.Methods: 1.K562 cell line was chose as the research object. 2.Five plasmids were constructed from our laboratory. The transfectants with pGenesil-2-Bmi-11, pGenesil-2-Bmi-12, pGenesil-2-Bmi-13, pGenesil-2-Bmi-14 and pGenesil-2-HK were named K562-S1, K562-S2, K562-S3, K562-S4 and K562-V cells, respectively. The parental K562 cells(wild-type) were named K562-W. 3. Bmi-1 RNA and protein after different plasmid transfection were detected by RT-PCR and Western blot. 4. K562 cells were treated with different plasmids at different time points, meanwhile, the cell viability was tested by MTT test. 5. Cell autophagy in each group were detected by transmission electron microscope scanning test. 6. Each group of various proteins including PTEN, Beclin1, LC3, Akt, p-Akt and mTOR expression were detected by Western blot.Results: 1. the Bmi-1 mRNA and protein were significantly decreased in K562-S1 and K562-S3 cells. So K562-S1 and K562-S3 cells were chosen to be used for the following test. 2. K562-S1 and K562-S3 cells grew significantly slower than K562-W or K562-V cells from the third day after seeding, and there was no significant difference between K562-V and K562-W cells. 3. There were no autophagosome in K562-W and K562-V cells, however some autophagosomes were appeared in K562-S1 and K562-S3 cells. 4. The expression of Beclin1 and LC3-II were up-regulated with the inhibition of Bmi-1. p-Akt and mTOR expression was down-regulated in K562-S1 and K562-S3 cells with the inhibition of Bmi-1. PTEN protein expression in K562-S1 and K562-S3 cells was more up-regulated with the inhibition of Bmi-1. Treatment of K562-S1 with 3-MA decreased the expression of Beclin1, LC3-II, while no effects were observed in K562-V cells. Treatment of K562-S1 cells with rapamycin increased the expression of Beclin1 and LC3-II proteins, and decreased the expression of mTOR, but similar effects were also observed in K562-V cells.Conclusion: Inhibition of Bmi-1 expression could induce autophagy. Bmi-1-siRNA might modulate the PTEN/Akt/mTOR pathway involved in the autophagy of K562 cells.