MiR-181a Suppresses Chronic Myeloid Leukemia Stem/progenitor Cells Via Regulation Of GATA6 And SERPINE1

Objective:This thesis aims to delineate the role of miR-181a in regulation of the growth and tyrosine kinase inhibitor(TKI)response of chronic myeloid leukemia(CML)stem/progenitor cells and its underlying mechanism.CML originates from normal hematopoietic stem cells aquring BCR-ABL fusion gene,although TKI(e.g.Imatinib mesylate,IM)aganist BCR-ABL has greatly improved the management of CML pateints,some of those still suffer from relapse and TKI resistance due to the fact that leukemic stem/progenitor cells are still existing.Thus the better understanding of the growth and survival mechanism of these cells are key to improve the management of this disease.Methods:(1)To isolate and purify the bone marrow cells from healthy donors and CML patients,and obtain CD34+ cells to assess the expression of miR-181a;(2)miR-181a was delivery in to K562 cells with lentiviral vector,and then the effects of miR-181a on cell growth and IM response were compared with control cells;(3)the effect of miR-181a on the in vitro growth of BaF3/BCR-ABL cells was studied;(4)4 groups of cells were prepared,namely BaF3(scramble),BaF3(miR-181a),BaF3(BCR-ABL+scramble)and BaF3(BCR-ABL+miR-181a)cells,and then they were injected into lethally irradiated Balb/C mice via tail vein.The mice were observed closely to study the effect of miR-181a on the transform ability of BCR-ABL;(5)Genetic profiling data compring control and miR-181a expressed K562 cells were generated,the down-regulated genes upon miR-181a expression were screened for potential miR-181a target genes with TargetScan software;(6)Both mRNA and protein expression of GATA6 and SERPINE1 was assessed in control and miR-181a expressed K562 cells;(7)The expression of both GATA6 and SERPINE1 in CD34+cells from healthy donors and CML pateints was analyzed with Q-RT-PCR.(8)The effects of GATA6 silencing and SERPINE1 silencing on the growth and IM response of CML cells were studied.Results:(1)The expression of miR-181a in CML CD34+cells was significantly reduced compared with normal control cells;(2)miR-181a inhibited the proliferation of K562 cells and enhanced their IM sensitivity;(3)miR-181a inhibited the in vitro growth of BaF3/BCR-ABL cells;(4)miR-181a attenuated BCR-ABL induced mice leukemia;(5)miR-181a inhibited both mRNA and protein expression of GATA6 and SERPINE1 in K562 cells,according to the indication of gene expression profile data and bioinformatics prediction;(6)The expression of both GATA6 and SERPINE1 in CML CD34+cells was significantly higher than that in normal control cells;(7)The silence of GATA6 or SERPINE1 inhibited the growth of K562 cells and enhance their IM sensitivity;(8)The silence of GATA6 or SERPINE 1 significantly inhibited the colony-forming cell ability of CML CD34+ cells(n=3).Conclusion:miR-181a was significantly down-regulated in CML CD34+cells;miR-181a inhibited the growth of CML cells and enhanced their TKI sensitivity partially through its regulation of both GATA6 and SERPINE1.