Effect Of Skp2 On Proliferation Of Chronic Myeloid Leukemia Cells And Its Mechanism Of Imatinib Sensitivity

BackgroundChronic myeloid leukemia（CML）is a malignant proliferative disease of bone marrow.It is mainly characterized by the production of a large number of immature leukocytes from bone marrow.Incidence rate of CML is 20%.At present BCR-ABLfusion gene is considered to be the key cause and important feature of CML.The expression of the fusion gene can produce BCR-ABL,a chimeric protein with constitutive tyrosine kinase activity.In the clinical treatment of CML,specific BCR-ABL inhibitors,such as tyrosine kinase inhibitors（TKI）,can significantly improve the survival rate of patients,and have been widely used in clinical.Imatinib is a kind of TKI that blocks the inactive conformation of BCR-ABL protein.It is first used in the treatment of chronic myeloid leukemia.It has significant effect and low toxicity.However,a large number of CML patients are not sensitive to imatinib or resistant to imatinib through a variety of cellular mechanisms.Therefore,it is very urgent to select effective therapeutic targets in the progress of CML,which will help to improve the chemotherapy effect of CML in imatinib treatment.S-phase kinase associated protein 2（Skp2）is E3 ubiquitin ligase,which is encoded by human Skp2 gene.Skp2 is a direct c-myc target gene,which plays an important role in the regulation of cell cycle.It is often overexpressed in a variety of human malignant tumors.In serum deficient cells,Skp2 overexpression induces cyclin A accumulation and p27 phosphorylation dependent degradation,which promotes the cell to enter S phase.Skp2 is also involved in ubiquitination of other cell cycle regulatory proteins,including cyclin E and transcription factor E2F1.At present,more and more evidences show that Skp2 is related to the drug resistance of anti-tumor drugs.For example,Skp2 positively regulates the expression of MAD2 through p27-CDKs-E2F1,and can enhance the sensitivity of lung cancer cells to paclitaxel by inhibiting Skp2.However,the role of Skp2 in CML and anti-tumor drug resistance remains to be determined.ObjectiveTo compare the protein and m RNA levels of Skp2 in peripheral blood leukocytes between CML patients and normal controls.To evaluate the mechanism of abnormal expression of Skp2 in K562 cells.Knock-down and overexpression of Skp2 gene in K562 cells,and detect the change of cell number and cell proliferation ability.Skp2m RNA was detected by knock-down and overexpression of c AMP response element binding protein（CREB）in K562 cells.The sensitivity of K562 cells to imatinib was detected after inhibition of PI3K/Akt-CREB-Skp2 axis.Research methods1.Collect 24 newly diagnosed CML patients（Study Group）and 7 healthy people（control group）samples.Collect the peripheral blood of the study group and the control group,and separate the leukocytes from the peripheral blood samples.The m RNA level of Skp2 was analyzed by real-time fluorescent quantitative PCR（QRT PCR）and Western blot was used to analyze the protein level of Skp2.2.Human CML cell line K562 was cultured in RPMI 1640 medium containing 10%fetal bovine serum at 37℃and 5%CO₂.Skp2 sh RNA plasmid based on plk0.1 was constructed.Skp2 was stably knocked down in K562 cells.Western blot was used to detect Skp2 protein level in K562 cells stably expressing sh RNA-ctrl,sh RNA-skp2-1 or sh RNA-skp2-2,and the effect of Skp2 gene knock-down was verified.The proliferation rate of K562 cells was compared under the condition of Skp2 stable knock-down and non knock-down by cell count analysis.The nuclei of K562 cells stably expressing sh RNA-Ctrl,sh RNA-skp2-1 or sh RNA-skp2-2were observed by Ed Ustaining and Hoechst 33342 staining,and the ratio of Ed U positive cells to Hoechst 33342 positive cells was detected.The Flag-Skp2 plasmid was constructed,and the protein levelof Skp2 in K562 cells with ectopic expression of Skp2 was determined by Western blot analysis andthe number of K562 cells was measured by cell count analysis.For K562cells with ectopic expression of flag or flag-Skp2,the nucleus was observed by Ed U staining and Hoechst 33342 staining,and the ratio of Ed U positive cells to Hoechst33342 positive cells was detected.3.Using Jas PAR software to detect the upstream genome sequence of Skp2 gene,a potential CREB binding region（BR）was found in Skp2 promoter.Chromatin immunoprecipitation（Ch IP）analysis was used to confirm that CREB gene was related to the gene fragment of CREB binding region（BR）in Skp2 gene.A series of wild-type CREB-BR（WT）p GL3 luciferase reporter constructs andmutant（MUT）p GL3 of missing CREB-BR were constructed.K562 cells were cotransfected with p GL3 based luciferase and flag or Flag-CREB plasmids containing wild-type or mutant Skp2promoter regions.After 24 hours of cotransfection,the lysate was taken and its transcriptional activity was measured by luciferase.Western blot was used to confirm the successful ectopic expression of CREB.Meanwhile,the m RNA level of Skp2 was measured by q RT-PCR.K562 cells were cotransfected with sh RNA-CREB gene or sh RNA-Ctrl gene.After 24 hours of cotransfection,the lysate was taken for luciferase analysis and Western blot to confirm the gene knock-down effect.The changes of Skp2m RNA in K562 cells before and after CREB gene knock-down were detected by q RT-PCR.4.K562 cells stably expressing sh RNA-Ctrl,sh RNA-Akt,sh RNA-CREB or sh RNA-skp2 were treated with 1μM imatinib for 24 hours.The cell viability was measured by MTT colorimetry and compared with sh RNA-Ctrl group.The relative cell viability was obtained by three experiments.K562 cells were pretreated with DMSO or phosphatidylinositol 3-kinase inhibitor（Ly29402）for 24 hours,and then treated with 1μm imatinib for 24 hours.The cell viability was measured by MTT colorimetry and compared with the control group.K562 cells stably expressing sh RNA-Ctrl,sh RNA-Akt,sh RNA-CREB or sh RNA-Skp2 were treated with 1μm imatinib for 24hours,and lysosomes were assayed for caspase 3/7 activity and PARP cleavage by Western blot analysis.K562 cells were pretreated with 1μm imatinib for 24 hours,and the activity of Caspase-3/7 was detected by kit and the cleavage of PARP was analyzed by Western blot.Results1.Compared with the healthy control group,Skp2 was abnormally expressed in CML patients.Peripheral blood leukocytes were collected from 24 CML patients and 7healthy people.CML samples showed that the m RNA and protein levels of Skp2 were significantly higher than those of the healthy control group.The data showed that Skp2was up-regulated abnormally in CML patients,and Skp2 protein level was consistent with m RNA level.2.Skp2 was the key to the proliferation of K562 cells.Skp2 gene was knocked down stably in K562 cells.Compared with the control cells,the number of cells knocked downof Skp2 was significantly reduced.Edu analysis showed that the proliferation of K562 cells with Skp2 knock-down was significantly lower than that of the control group.3.Skp2 overexpression had an effect on the proliferation of K562 cells.Contrary to the knock-down results,the number of K562 cells increased by ectopic expression of Skp2gene.Related to this,over expression of Skp2 resulted in a significant increase in the percentage of Edu positive cells in K562 cells.4.Skp2 was regulated by CREB transcription.A putative CREB binding region（BR）was found in the Skp2 promoter.Chromatin immunoprecipitation（Ch IP）analysis showed that CREB was associated with the gene fragment of CREB binding region（BR）in Skp2 gene.A series of wild-type CREB-BR（WT）p GL3 luciferase reporter constructs and mutant（MUT）p GL3 of missing CREB-BR were constructed.Skp2promoter with WT or MUT and plasmid with flag or flag-CREB were cotransfected into K562 cells.The transcription activity of wild-type CREB-BR（WT）increased significantly after overexpression of CREB,but MUT and p GL3 were empty There was no significant increase in body transcription activity.On the contrary,the transcriptional activity of wild-type CREB-BR（WT）was decreased in K562 cells stably knock-down CREB gene,and no change of transcriptional activity was found in MUT and p GL3.5.The knock-down of CREB resulted in a significant decrease of Skp2 m RNA level in K562 cells.In view of the fact that transcriptional activity of CREB was induced by phosphorylation of its 133 serine,we tried to evaluate the effect of wild-type Flag-CREB and mutant Flag-CREB-Ser-133Aon Skp2 expression.The wild-type CREB can up regulate the m RNA level of Skp2,but the mutant CREBS 133A has no obvious abnormal regulation on the m RNA level of Skp2.These results suggest that the expression of Skp2 is regulated by CREB at the transcription level in K562 cells.6.Inhibition of PI3K/Akt-CREB-Skp2axis increased the sensitivity of K562 cells to imatinib.K562 cells stably knocked down by CREB or Skp2 showed a sharp decline in cell viability after treatment with imatinib.7.The knock-down of CREB or Skp2 resulted in a significant increase of Caspase-3/7activity in imatinib treated K562 cells.The activation of Caspase-3/7 was in response to imatinib treatment.In addition,the percentage of PARP and caspase-3 cleavage of K562cells stably knocked down by CREB or Skp2 after imatinib treatment was higher than that of the control cells after drug treatment.8.PI3K/Akt signaling pathway plays a specific role in the activation of CREB and the expression of Skp2.The stable knock-down of Akt and the inhibition of PI3K/Akt pathway by LY294002 in K562 cells can significantly reduce the activity of K562 cells treated by imatinib,andresulted in the decrease of Skp2 protein level,the increase of Caspase-3/7 activity and the PARP cleavage,which indicated that the sensitivity to imatinib was enhanced.ConclusionCompared with the healthy controls,the high expression of Skp2 in the peripheral blood leukocytes of Initial treatment of CML patients is very important for the proliferation of CML cells.In mechanism,Skp2 is transcriptional regulated by PI3K/Akt CREB signaling pathway in K562 cells.In addition,the sensitivity of K562 cells to imatinib could be significantly increased by stably knocking down the expression of Skp2 or blocking the PI3K/Akt-CREB pathway.