The Regulatory Roles Of MiR-17-92 In Leukemogenesis Of Chronic Myeloid Leukemia And Its Mechanism

Chronic myelogenous leukemia(CML)is a hematological malignancy derived from the hematopoietic stem cell malignant transformation caused by BCR-ABL fusion oncogene.BCR-ABL fusion gene encoding the BCR-ABL fusion protein promotes tyrosine kinase(TK)overexpression,and TK mainly catalyzes the acidification of protein tyrosine enzyme to activate the multiple cellular pathways,resulting in accelerating cell proliferation and inhibiting apoptosis.Currently,CML therapeutics strategies include the bone marrow transplantation,gene therapy,immune therapy and molecular target therapy,especially tyrosine kinase inhibitors(TKI).TKI is specific inhibitor to block BCR-ABL mediated malignant transformation,moreover it has low toxicity and less damaged effect on normal cells.TKIs have been widely used in clinical for treatment of hematopoietic malignancies.However,the drug resistance and relapse from TKI insensitivity of leukemia stem cells are still the major obstacles to cure CML.It is necessary to extensively explore the biological basis of CML leukemia stem cells and the molecular mechanisms of TKI resistance to identify new therapeutic targets.Micro RNAs(miRNA)are a non-coding small molecule single-stranded RNA with 21nt-23 nt length.It mainly post-transcriptionally regulates the expression of target genes and involves in cell proliferation,differentiation and apoptosis,and even the initiation of tumors.miRNAs regulate tumorigenesis mainly through the tumor target molecular recognition,miRNAs mutation and as oncogenes or tumor suppressor genes.It has reported that numerous miRNAs participate in the transformation of hematopoietic stem cells.The upregulation or downregulation of miRNA affect the progress and development of CML by relating target genes and signaling pathways.MiR-17-92 clusters also called oncomir-1 locating in 13q31.3 has mutations and amplification in hematological cancer and the myeloma.Our previous works have shown that the expression of miR-17,miR-18 a,miR-19 a,miR-20 a,miR-19 b,and miR-92 six mature miRNAs increases in CML CD34 + cells detected by gene chip and Taqman Q-PCR.However,the roles of miR-17-92 in pathogenesis of CML and its mechanisms remain unclear.This study will establish BCR-ABL induced CML model by using miR-17-92 gene conditional knockout mice in order to evaluate the function and mechanism of miR-17-92 in CML.Firstly,we clarified that miR-17-92 expression induced by BCR-ABL activation was mediated by c-Myc pathway by using si RNA silenced strategy in CML cells.The c-Myc si RNA were transduced into CML cell line KCL22 cells by using Lipofectamine? 3000.The c-Myc expression was detected by Q-PCR method,and miR-17-92 expression was identified by Taqman Q-PCR.The Q-PCR data showed that the expression of miR-17?miR-18a?miR-19a?miR-20a?miR-19 b was reduced in c-Myc silenced CML cells.It is suggested that c-Myc mediated the upregulation of miR-17-92 expression induced by BCR-ABL.Secondly,the regulatory roles of miR-17-92 in proliferation and survival of CML cells were further determined.The K562 cells was transfected with inhibitors of miR-17?miR-18a?miR-19a?miR-20a?miR-19 b and miR-92.The cell proliferation was detected by using the CCK-8 assay kit and the apoptosis was determined by Annexin V-APC/PI staining followed measured by flow cytometry.The results showed that miR-17-92 inhibitors decreased cell proliferation and increased apoptosis in CML cells.Thus,miR-17-92 is an oncogene in CML.Thirdly,we examined the role of miR-17-92 in BCR-ABL–induced leukemogenesis and development using transplantation of miR-17-92 deficient bone marrow cells transduced with retrovirus(P210)carrying BCR-ABL fusion gene.The conditional deletion efficiency of miR-17-92 cluster in bone marrow cells of knockout mice were detected by Q-PCR.The expression of miR-17,miR-18 a,miR-19 a,miR-20 a,miR-19 b and miR-92 in knockout mice was significantly lower than those in wild type(WT)mice.The knockout mice demonstrated that miR-17-92 is essential for B cells generation without affecting the myeloid hematopoiesis.Male WT mice and miR-17-92 knockout mice as donors were primed by intravenous injection with 5-fluorouracil(5-FU).Bone marrow cells of two donor groups were harvested,and stimulated with hematopoietic growth factors and transduced with P210 BCR-ABL retrovirus.These transduced cells were transplanted into lethally irradiated wild type recipient mice via tail vein injection.After transplantation,the recipient mice were recorded the survival and evaluated daily for signs of morbidity,weight loss,failure to thrive,and splenomegaly.The hematopoietic tissues and cells were of these mice were analyzed by using multiple assays,including weight record,flow cytometry analysis,histopathology and genomic DNA preparation.The results showed significant differences in two groups,one is mice transplanted with P210 transduced bone marrow cells of miR-17-92 knockout mice(K group)and another one is mice transplanted with P210 transduced bone marow cells of normal WT mice(N group).Significantly,all mice that received a transplant of P210 infected cells developed a CML-like disease and showed a high percentage of GFP+ cells in their peripheral blood within 30 days.However,the percentage of GFP+ cells in K group was lower than that of N group.The increase of peripheral blood cells occurs earlier in N group than that of K group.It is interesting to note that the spleen size of K group was obviously bigger than that in N group.The structure of hepatic lobule was not complete with crowded liver beam,and lymphoid tissue like nests diffused in structure,and lymphoid cells were round or ovoid with large nuclear and less pulp.The spleen tissues had more white pulp and less red pulp with some white blood cells.The most important data were that some of the mice in K group showed a substantially increased survival rate,with more than 30% alive at 70 days after bone marrow transplantation(BMT),whereas the WT group died within 40 days post BMT.This suggests that loss of miR-17-92 function could inhibit the oncogenic transformation activity of BCR-ABL in CML.Finally,the mechanisms that miR-17-92 regulates leukemogenesis were further clarified.We identified that A20(TNFAIP3)functions as a target of miR-19 b in CML cells.Q-PCR data showed A20 expression in the bone marrow mononuclear cells of K group increased compared to that of N group.A20 was predicted to have potential miRNA sites such as miR-18 a,miR-19 a and miR-19 b in its 3'-UTR by Target Scan software.After appropriate annealing,digestion and ligation,the products were cloned into the Nde I and Xba I sites on the PGL-3M vector downstream of the luciferase gene to generate p GL3-A20-miR-18 and p GL3-A20-miR-19a/b construct.The constructs were respectively co-transfected with miR-18 a,miR-19 a and miR-19 b mimic into 293 cells.The dual luciferase reporter gene indicated that luciferase activity of A20 3'UTR-miR-19 b group was significantly lower than that of the control group.WB assay demonstrated that expression of A20 increased in both K562 and KCL22 cells treated by miR-19 b inhibitor or Imatinib,conversely the expression of A20 was decreased in cells transfected with miR-17-92 overexpression lentivirus.Q-PCR data showed the expression of A20 in CML CD34+ cells was lower than that in normal donors,whereas the A20 expression was increased after CML CD34+ cells were treated by Imatinib.These results indicated that A20 downregulation in CML cells depends on BCR-ABL activity.The roles of A20 in regulation of proliferation and survival of CML cells were investigated.A20 overexpression lentivirus was packaged by the calcium phosphate precipitation technique.Transduction of lentivirus A20 results in A20 expression in K562,KCL22 and CML CD34 cells determined by Q-PCR and WB.After CML CD34 cells were transfected by A20 overexpression lentivirus,the cell apoptosis,cycle and proliferation was detected by flow cytometry,and the ability to form colonies of BFU-E,CFU-GM and CFU-GEMM were evaluated in vitro.A20 overexpression inhibited the proliferation and colony formation,increased G0/G1 phase of the cell cycle,decreased the proportion in S phase,and promoted cell apoptosis of CML CD34+ cells.Thereby,A20 may function as a tumor suppressor gene in CML.In order to explore the mechanism that A20 affects the characteristics of CML cells,we checked the P65 and I?B? expression by WB in CML cells transfected by A20 and miR-17-92 overexpression lentivirus.A20 overexpression decreased the phosphorylation expression of P65 and I?B? in K562 and CML CD34 cells,whereas miR-17-92 overexpression increased the phosphorylation expression of P65 and I?B? in K652 cells.However,there was no difference in KCL22 cells.Therefore,A20 inhibits P65 and I?B? phosphorylation resulting in negatively regulating NF-?B signaling pathway.In conclusion,our studies demonstrated that miR-17-92 is a critical contributor to CML leukemogenesis.The innovation of this study is that it clarified a mechanism that miR-17-92 promotes leukemogenesis and regulates drug resistance in chronic myeloid leukemia via targeting A20 and activation of NF-?B signaling.It raises the possibility of targeting miR-17-92 regulated pathways to improve therapeutic outcomes of CML.