| Objective: Leukemia is a malignant cloning disease derived from hematopoietic stem cell,is one of the most common human malignancies in adolescents and young adults.but multi-drug resistance(MDR)will eventually result in failure of chemotherapy in patients.New research suggested a proposed mechanisms for MDR is the presence of leukemia stem cell(LSCs),because Stem cells will be relative rest state in body under a normal condition,and have a strong apoptotic resistance and DNA damage repair capacity.Bmi-1 is a self-renewal factor of stem cells,which has been reported to be over-expressed in various malignant tumor cell lines and regulated cellular processes including cell cycle progression,apoptosis and senescence as well as immortalization.In our study,we choose the Chronic myelogenous leukemia(CML)K562 cell line to study if Bmi-1 gene is involved in the self-renew of K562 cells and further to explore its possible molecular mechanisms.Methods: 1.leukemia cell line K562 cells were chose as the researching object.K562 cells were engineered to be silenced for Bmi-1 expression using RNA interfering method by transient transfecting transfectants 48 hours,and obtained the K562-S1 and K562-S3 cells and compare the expression of Bmi-1 between the two cells with parental K562 cells by western and RT-PCR.2.Soft agar cloning assay and Tumor xenograft in nude mice were conducted to test the self-renewal ability of K562 cells when the Bmi-1 expression were reduced.3.Examine the expression of PTEN,P-AKT,AKT between K562,K562-S1 cells via western blot.4.Select 20 uM LY294002(PI3K/AKT inhibitor)to treat the K562 cells for 36 hours and obtain the K562-LY cells to conduct soft agar cloning assay and Tumor xenograft in nude mice.5.Select 120 nM Bpv(PTEN inhibitor)to treat the K562-S1 cells for two hours and obtain K562-S1-B3 cells,then western blot were performed to examine the change of Bmi-1,PTEN,P-AKT and AKT expression.Soft agar cloning and tumor xenograft in nude mice were conducted to compare the self-renewal ability between K562,K562-S1 and K562-S1-B3.6.We evaluated these subcutaneous tumors from nude mouse by immunohistochemistry for Bmi-1 and ki-67 expression.Results: 1.the Bmi-1 mRNA and protein were significantly decreased in K562-S1 and K562-S3 cells.2.The clonogenicity in soft agar and tumorigenicity in nude mice of K562-S1 and K562-S3 cells significantly reduced than K562 or K562-CTR cells,and there was no significant difference between K562-CTR and K562 cells.K562-S1 cells were chosen to be used for the following test because of it's better effect.3.Western results showed that when the K562 cells were transfected with Bmi-1 siRNA,the expression of PTEN protein was increased,P-AKT protein expression was decreased.4.When the P-AKT expression was suppressed in K562-LY cells,the clonogenicity and tumorigenicity of K562-LY group was decreased.5.When the PTEN was suppressed in K562-S1 cells,P-AKT expression was up-regulated,there was no change in the total amount of AKT expression.But soft agar cloning assay and Tumor xenograft show that in comparison with the control cells,the clonogenicity and tumorigenicity treated with Bmi-1-siRNA were significant suppressed.While the K562-S1 cells were treated with PTEN inhibitor 120 nM Bpv,the clonogenicity and tumorigenicity were increased.6.The result of immunohistochemistry indicate that Bmi-1-siRNA dramatically suppressed the Bmi-1and ki-67 protein levels in xenograft tumor tissue.But when we treated the K562-S1 cells with PTEN inhibitor Bpv,the Growing tumors indicated an increase in Bmi-1 and ki-67 expression.Notably,the Bmi-1 and ki-67 expression were decreased in PI3K/AKT inhibitor groups compared with K562 groups.Conclusion: Bmi-1 could mediate the clonogenicity and tumorigenicity of K562 cell,and PI3K/AKT is one of the important pathways. |
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