The Function And Mechanism Of MiR-181a/SERPINE1 Axis In Chronic Myeloid Leukemia Stem/Progenitor Cells

Chronic myeloid leukemia(CML)is a hematological malignancy originating from hematopoietic stem cells.The annual incidence is about 1/100,000～2/100,000,accounting for 1/6 of the total cases of leukemia in adults.More than 95%of CML patients bear BCR/ABL fusion gene,which encodes a tyrosine kinase with constitutive activity to induce CML.Tyrosine kinase inhibitors(TKIs)has significantly extended patient survival period,however,a few CML patients achieve disease free survival after drug withdrawal,and some patients still have the risk of relapse and drug resistance.TKIs primarily target differentiated cells but not leukemic stem cells(LSCs),suggesting there are some other regulatory pathways independent to BCR/ABL responsible for the survival of LSCs,which prompts researchers to focus on the mechanism of survival and self-renewal of LSCs,and to identify new targets to eliminate LSCs.Accumulating evidences support that micro RNA is implicated in the survival of CML stem cells,the delineation of its function and mechanism will deepen the understanding of the molecular pathogenesis of diseases and provide new strategies to improve the treatment of diseases.Objective:This thesis aims to investigate the functions and mechanisms of the miR-181a/SERPINE1 axis in CML stem/progenitor cells,and to identify potential new targets for CML therapy,and to provide new strategies for improving the treatment of CML.Methods:(1)Using RT-qPCR to detect the expression level of miR-181a in CML CD34~+stem/progenitor cells compared with normal CD34~+stem/progenitor cells,and with TKI treatment.(2)CFC assay to detect the effect of miR-181a overexpression on growth and Imatinib sensitivity of CML CD34~+stem/progenitor cells.(3)Using BCR/ABL transformed Ba F3 cells to construct leukemia mice model,the growth and invasion of miR-181a overexpression BCR/ABL-Ba F3 in vivo was investigated.(4)Bioinformatics analysis to screen the candidate targets of miR-181a in CML stem/progenitor cells.(5)RT-qPCR,western blot and dule luciferase reporter analysis were utilized to confirm whether SERPINE1 was a new target m RNA of miR-181a.(6)CFC assay to detect the effect of SERPINE1 silencing on the growth and Imatinib sensitivity of CML CD34~+stem/progenitor cells.(7)Overexpression of SERPINE1 to investigate whether SERPINE1 could rescue the growth inhibition and Imatinib sensitization of miR-181a overexpression on CML stem/progenitor cells.(8)CCK-8 was used to study the effect of SERPINE1 inhibitors on CML cells with Graphpad to calculate IC50.(9)CFC assay investigated the effect of SERPINE1 inhibitor on the growth and Imatinib sensitivity of CML CD34~+stem/progenitor cells.(10)Annexin V/PI and western blot were used to detect the apoptosis of CD34~+stem/progenitor cell upon SERPINE1 inhibitor treatment.(11)JC-1,Mito-tracker Red CMXRos staining and ROS detection were used to study the effects of SERPINE1 inhibitor on mitochondrial activity of CML cells.Results:(1)Compared with normal bone marrow,miR-181a had lower expressed level in CML CD34~+stem/progenitor cells,and miR-181a expression was activated in CML cells upon TKI treatment.(2)Overexpression of miR-181a significantly inhibited the growth of CML CD34~+stem/progenitor cells and enhanced its sensitivity to Imatinib.(3)miR-181a significantly inhibited the leukemogenesis of BCR/ABL transformed Ba F3 in vivo.(4)Bioinformatics analysis suggested that SERPINE1 was a potential target m RNA of miR-181a.(5)RT-qPCR,Western Blot and dule luciferase reporter analysis confirmed that miR-181a directly regulated the expression of SERPINE1 m RNA through motif-2 domain on its3’UTR.(6)SERPINE1 silencing significantly inhibited the growth of CML CD34~+stem/progenitor cells and enhanced their Imatinib sensitivity.(7)Overexpression of SERPINE1"rescued"the growth inhibition and Imatinib sensitization of miR-181a overexpression in CML cells.(8)SERPINE1 inhibitors,Tiplaxtinin and TM5441significantly inhibited the proliferation of CML cells.(9)Tiplaxtinin significantly inhibited the CFC production of CML CD34~+stem/progenitor cells and enhanced their Imatinib sensitivity,while had little toxicity to normal CD34~+stem/progenitor cells.(10)Tiplaxtinin effectively induced caspase-dependent apoptosis of CML CD34~+stem/progenitor cells.(11)Tiplaxtinin significantly increased ROS,while decreased mitochondrial activity and depolarization in CML cells.Conclusion:We found that miR-181a had aberrant expression in CML stem/progenitor cells,and identified a novel miR-181a/SERPINE1 axis to regulate the growth and IM response of CML stem/progenitor cells.Mi R-181a suppressed the proliferation of CML stem/progenitor cells in vitro and inhibited leukemogenesis in vivo;in addition,it enhanced the sensitivity of CML stem/progenitor cells to TKI treatment through its suppression of SERPINE1.Targeting SERPINE1(RNAi or inhibitor)strongly inhibited the growth of CML cells,SERPINE1 inhibitor alone or in combination with TKIs effectively induced apoptosis of CML stem/progenitor cell but not normal control cells,which demonstrated that SERPINE1 could be a novel therapeutic target for CML treatment.