MiR-34c-5p Promotes Eradication Of Acute Myeloid Leukemic Stem Cells By Inducing Senescence Via Selectively Targeting RAB27B To Inhibit Exosomes Shedding

Part I The level of miR-34c-5p in acute myeloid leukemia stem cells and its correlation with prognosis and chemotherapeutic effect of AML patients Objective: In a pathway analysis of senescence-associated miRNA,researchers found miR-34 c had the potential to serve as a key regulator of all senescence induction pathways.Our pre-experiments found that there existed very low expression of miR-34c-5p in leukemia stem cells(LSCs).In this study we aimed at expanding the clinical sample size and further validating the result,estabolishing a link between the association of the level of miR-34c-5p in AML stem cells and its effects on the prognosis and chemotherapeutic reaction of AML patients.Methods: CD34+CD38-cells were isolated from bone marrow(BM)of primary AML patients and normal volunteers,human cord blood(h CB)and AML cell lines by following the instructions of CD38 and CD34 Micro Bead Kits.The expressions of miR-34c-5p in different samples derived CD34+CD38-cells were detected by quantitative Polymerase Chain Reaction(q PCR).Then AML patients were classified into better,intermediate and poor prognosis groups according to National Cancer Center Network(NCCN)guidelines,the correlation between miR-34c-5p expression level and AML prognosis was analyzed;the patients under a unified therapeutic regimen of daunorubicin and cytarabine(DA)were followed-up and classified into Complete Remission(CR)and Not Complete Remission(NCR)according to their therapeutic efficacy,the correlation between miR-34c-5p level and AML therapeutic efficacy was analyzed.Results: A significantly lower level of miR-34c-5p was discovered in LSCs from AML patients in the poor and intermediate prognosis groups compared with that in the better prognosis group and normal control group.Moreover,a higher miR-34c-5p level in AML CD34+CD38-cells corresponded to a better therapeutic efficacy for AML patients.Conclusions: miR-34c-5p is down-regulated in primary AML CD34+CD38-cells and is associated with adverse prognosis and poor therapeutic efficacy in AML patients.Part II Increased miR-34c-5p induces AML stem cells senescence and promotes eradication of AML stem cells in immune deficient mice Objective: In the first part,we have demonstrated that miR-34c-5p is down-regulated in AML stem cells.So,in this part,our aims were to investigate:(1)the effects of the increased miR-34c-5p on cell proliferation,cell cycle,apoptosis and senescence of AML stem cells;(2)whether the enhanced miR-34c-5p expression could prevent the reestablishing of leukemia model by LSCs and promote the eradication of LSCs in NOD/SCID or NOG mice.Methods:(1)We transfected miR-34c-5p mimic into AML stem cells through electroporation.72 h later,cells were collected;cell proliferation,cell cycle,cell apoptosis and senescence were analyzed by flow cytometry.Cell cycle associated genes and senescence associated secretory phenotype(SASP)were detected by q PCR and western blotting;(2)KG-1a cells were transduced with lentiviruses that were empty(CTRL)or overexpressed miR-34c-5p(34c OE)and then cultured with puromycin for positive selection of lentivirus-transfected cells until the GFP+ population of the CTRL and 34 c OE groups reached more than 90%,the sublethal dose irradiated NOG mice were transplanted with 8×106 KG-1a cells transduced with control(CTRL)lentivirus or miR-34c-5p overexpression(34c OE)lentivirus.At 9 weeks post-transplantation,the mice were euthanized and the percentage of h CD45+,or h CD45+CD33+ or h CD45+CD34+ cells from BM,spleen,liver,lung,peripheral blood were assessed using flow cytometry.Then For serial transplantation,the sublethal dose irradiated NOD/SCID mice were transplanted with 8×106 CTRL-KG-1a or 34 c OE-KG-1a cells and euthanized at 7 weeks post-transplantation,the percentage of h CD34+CD38-or h CD34+ cells in the BM and spleen were analyzed by flow cytometry,Then,6.3×105 h CD34+ AML cells from each recipient mouse were secondarily transplanted into another sublethally irradiated NOD/SCID mouse.At 7 weeks after transplantation,BM cells were harvested from the second transplanted mice,and h CD45+CD34+ % was detected by flow cytometry.Results:(1)We found that the forced expression of miR-34c-5p resulted in a decreased cell number,proliferation index and percentage of Ed U+ cells,induced an increase in the number of G0/G1 phase cells and decreased cell cycle-associated protein levels;up-regulation of miR-34c-5p failed to induce apoptosis but resulted in significantly increased senescence in AML stem cells.(2)The NOG mice injected with 34 c OE-KG-1a cells presented significantly decreased h CD45+,or h CD45+CD33+ or h CD45+CD34+ cells from BM,spleen,liver,lung,and peripheral blood compared with that in the CTRL group.The NOD/SCID mice injected with 34 c OE-KG-1a cells presented significantly decreased h CD34+CD38-and h CD34+ cells in the BM and spleen;h CD45+CD34+% in BM of the second transplanted mice was significantly attenuated in the 34 c OE group compared with that in the CTRL group.Conclusions:(1)Increased miR-34c-5p induces senescence of AML CD34+CD38-cells ex vivo.(2)Increased miR-34c-5p expression prevents leukemia development and promotes eradication of AML stem cells in immune deficient mice.Part III The mechanisms of AML stem cells senescence resistance induced by the low level of miR-34c-5p Objective:In this part,we investigated the mechanisms of miR-34c-5p induced AML stem cells senescence and miR-34c-5p deficiency in AML stem cells.Methods:(1)The transcriptome difference was analyzed between CTRL-KG-1a and 34 c OE-KG-1a cells.The expression of cell cycle-and senescence-associated genes selected from the sequence data were further confirmed by q PCR and western blotting.(2)KG-1a cells and their culture supernatant were harvested at different time points and the miR-34c-5p expression was assessed via q PCR.(3)After KG-1a cells were treated with vehicle or 1.0 ?M GW4869 for 48 h,miR-34c-5p expression in KG-1a cells,apoptotic bodies(AB),microvesicles(MV)and exosomes(Exo)was detected by q PCR.(4)q PCR,western blotting and a luciferase reporter assay were performed to analyze whether miR-34c-5p targeting RAB27B;(5)Transfected KG-1a cells with RAB27B-siRNA to analyze whether RAB27 B regulating exosomes-shedding of miR-34c-5p? Results:(1)Transcriptome sequencing showed that compared to CTRL-KG-1a cells,senescence-associated genes P21,P53,PAI-1,IL8,MMP2/9,53BP1,H2 AX and DEC1 etc was increased to some degree in 34 c OE-KG-1a cells except for p16 and SIRT1.But cell cycle-associated genes,including CDK1/4/6,Cyclin D/E,E2F3,c-Met and P27 had decreased expression in 34 c OE-KG-1a cells;the selected genes from the transcriptome sequence data set were further validated by q PCR and western blotting.(2)At different time points of KG-1a cell culturing,miR-34c-5p maintained a low expression in KG-1a cells but an increased level in culture supernatant.GW4869 could effectively increased miR-34c-5p in KG-1a cells but decreased miR-34c-5p in apoptotic bodies,microvesicles and exosomes from KG-1a culture supernatant.(3)Transcriptome sequencing showed that among the genes differentially expressed in 34 c OE-KG-1a and CTRL-KG-1a cells,RAB27 B,an exosome-release regulatory gene,was abundantly expressed in CTRL-KG-1a cells and significantly down-regulated in 34 c OE-KG-1a cells;we further confirmed that RAB27 B m RNA and protein levels were decreased in 34 c OE-KG-1a cells by q PCR and western blotting;luciferase report assay further confirmed that miR-34c-5p is a specific regulator of RAB27 B.(4)Both single transfection of RAB27B-siRNA and co-transfection of both miR-34 c and RAB27B-siRNA(34c OE+RAB-Si)significantly decreased RAB27 B expression but increased miR-34c-5p expression;34c OE or RAB27B-siRNA significantly decreased miR-34c-5p levels in Exo but not in AB or MV;The level of the miR-34c-5p precursor(pre-miR-34c),which can directly reflect miR-34c-5p synthesis,was affected by RAB27B-siRNA.Conclusions:(1)miR-34c-5p induces senescence in AML CD34+CD38-cells through p53-p21Cip1-CDK/Cyclin or p53-independent CDK/Cyclin pathways.(2)miR-34c-5p directly targets RAB27 B to reduce exosome secretion-mediated miR-34c-5p trafficking in AML CD34+CD38-cells.