The Role Of Histone Demethylase RBP2 And MiR-370 In Blast Crisis Of Chronic Myeloid Leukemia And The Relative Mechanism

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by BCR/ABL fusion gene. The annual incidence is 1.0～1.5 per 100,000 persons. CML is exceedingly rare in children. In western world, the median age of CML patients is 50-60 years. Symptoms of CML include weight loss, weakness, sweats, bleeding, hypersplenotrophy, anemia, etc. The disease is triphasic, including an initial chronic phase (CP), an accelerated phase (AP), and finally a blastic phase (BP). More than 90 percent of CML patients are diagnosed as chronic phase, a relative early phase of the disease. Now CML-CP patients are sensitive to tyrosine kinase inhibitors (TKI), such as Imatinib and second-generation TKI.However, there are still 20 percent of CML-CP patients insentitive to both Imatinib and second-generation TKI, who spontaneously progress into blastic phase. Once the disease progresses into blastic phase, any kinds of treatment do not work, the fatality rate is extremely high. The blastic transformation of chronic myeloid leukemia is a transition of chronic myeloid leukemia into acute myeloid leukemia, which is the most common phenomenon in end-stage of chronic myeloid leukemia. The median lifetime of CML-BP patients is only 6 months. CML-BP patients are insensitive to any kinds of treatments, including the conventional combination chemotherapy, INF-α treatment, TKIs, etc. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only effective pathway to treat CML-BP patients. However, many problems still exist so that allo-HSCT can not be used widely, such as the low successful matching probability, GVHD related side effect, the high treatment costs and poor tolerance of the aged, etc. Therefore, revealing the intrinsic mechanism of CML-BP and looking for the new target is the key point. The existing research suggest that the intrinsic mechanism consists of BCR-ABL dependent and BCR-ABL independent mechanism. BCR-ABL fusion gene initiates CML, and it also plays an important role in CML progression. However, more and more studies suggest that BCR-ABL independent factors also promotes the blastic transformation of chronic myeloid leukemia, including transcription factor and epigenetic modifiers. Thus, a complex molecular regulatory network may exist in CML progression. Finding the key molecules is the basis of studying the intrinsic mechanism of CML-BP and looking for the new treatment targets.Epigenetics means heritable changes in gene expression without concomitant alteration in the DNA sequence. Epigenetic modifications consist of DNA methylation, histone modifications, and non-coding RNAs, etc, which are involved in regulating lots of cell biological processes, including cell proliferation, cell differentiation, and cell apoptosis, etc. The imbalance of epigenetic modifications play important roles in the development and progression of various tumors by upregulating the expression of oncogenes or downregulating the expression of tumor suppressor genes. The current studies suggest that DNA methylation and histone modifications are indeed involved in the blastic transformation of chronic myeloid leukemia. However, whether histone demethylase and non-coding RNAs play key roles in blast crisis transition remains largely unknown. Therefore, in this study, we choose histone demethylase RBP2 and non-coding RNA-miR-370 as the point. Through detecting the expression of RBP2 and miR-370 in CML-BP and studying their functions in leukemia cell lines, we provide a theory basis for finding new targets, which will be used for checking and treating the blast crisis in chronic myeloid leukemia.Part 1 Histone demethylase RBP2 decreases miR-21 in blast crisis of chronic myeloid leukemiaHistone demethylase Retinoblastoma binding protein 2 (RBP2), belonging to the JARID1 family proteins, specifically demethylates tri- and dimethylated lysine 4 of histone 3 (H3K4). RBP2 regulates lots of gene expression and determines cell fate.RBP2 affects many kinds of biological effects, such as development, proliferation, differentiation, senescence, angiogenesis, EMT, and circadian rhythms, etc. Defeo-Jones D etc originally found RBP2 could interact with pRb, then lots of studies find RBP2 can interact with many other proteins, such as p107、RBTN-2 PRC2、Mad、RBP-J、RunX2、 CLOCK-BMAL1.G9a, etc. Klose RJ etc firstly showed RBP2 was a histone demethylase in 2007. Misregulation of RBP2 is involved in many kinds of human cancers, such as breast cancer, gastric cancer, lung cancer, liver cancer, etc. However, leukemia is different from these solid tumor. Whether RBP2 plays an important role in blastic transformation of chronic myeloid leukemia is still unknown.Purpose:In order to study the role of RBP2 in blast crisis of chronic myeloid leukemia and the relative mechanism, we determine RBP2 expression in CML-CP and CML-BP samples, study the effects of RBP2 on differentiation and proliferation, and the regulatory mechanism between RBP2 and miR-21.Methods:(1)Detect the expression of RBP2 in CML-CP and CML-BP samples by qRT-PCR and Immunostaining.(2) Examine the effects of RBP2 on differentiation and proliferation in leukemia cellsAll experiments are designed in BCR-ABL(+) K562 cells and BCR-ABL (-) HL60 cells. After K562 and HL60 cells underwent granulocytic differentiation induced by DMSO or ATRA, the expression of RBP2 and differentiation-related genes (c-myc, Notchl, PU.1) were detected by qRT-PCR and Western Blot. Through transfecting RBP2 expression plasmid into K562 and HL60 cells, the effect of RBP2 on proliferation was studied by Cell proliferation assay and Soft agar assay of colony-formation.(3) Study the mechanism of RBP2 in CML progression① RBP2 negatively regulates miR-21After transfecting RBP2 expression plasmid into K562 cells, targeted miRNAs were screened by microRNA assay. According to microRNA assay and qRT-PCR, miR-21 might be a target gene of RBP2. Luciferase reporter assay was used to detect the change of miR-21 wide/mutant promoter activity. Whether RBP2 could directly bind to miR-21 promoter was proved by EMSA and CHIP.② RBP2 promotes PDCD4 expression by inhibiting miR-21After transfecting miR-21 mimics/inhibitor into K562 and HL60 cells, qRT-PCR and Western Blot were used to determine the level of PDCD4 mRNA and protein. In leukemia cells transfected with RBP2 expression plasmid or cotransfected with miR-21 mimics and RBP2 expression plasmid, PDCD4 expression and cell proliferation were different between two groups, and the difference were determined by qRT-PCR, Western Blot, Cell proliferation assay and Soft agar assay of colony-formation.Results:(1)RBP2 is underexpressed in CML-BPIn 26 CML-CP samples and 18 CML-BP samples, RBP2 expression was significantly reduced in CML-BP samples compared to CML-CP samples.(2) RBP2 induces leukemia cell differentiationWhen leukemia cells underwent granulocytic differentiation induced by DMSO or ATRA, RBP2 mRNA and protein were significantly upregulated. The expression of differentiation-related genes were also changed.c-myc and Notchl were downregulated and PU.1 was upregulated. The similar result was got in CML-BP primary cells.(3) RBP2 overexpression inhibits cell proliferationCell proliferation assay and Soft agar assay of colony-formation showed that K562 and HL60 cells transfected with RBP2 expression plasmid proliferated at a slower rate than with vector transfection, and their colony-formation ability was impaired.(4) In leukemia cells and CML primary cells, RBP2 downregulates miR-21 expressionRBP2 expression plasmid was transfected into leukemia cell lines and CML primary cells, and qRT-PCR showed that miR-21 expression was inhibited by RBP2 overexpression.(5) RBP2 binds to miR-21 promoter to repress its expression by demethylating H3K4 methylationLuciferase reporter assay suggested that RBP2 inhibited miR-21 wide promoter activity. After the binding site was mutated, miR-21 promoter activity had no change with RBP2 overexpression in leukemia cells.EMSA and CHIP confirmed that RBP2 bound to miR-21 promoter directly. Accompanied with RBP2 overexpession, the association between RBP2 and miR-21 promoter region was increased, and H3K4 trimethylation and dimethylation at the proximal promoter region of miR-21 were reduced. Besides, the whole level of H3K4 trimethylation and dimethylation was decreased.(6) RBP2 induces cell differentiation and inhibits cell proliferation depending on miR-21In leukemia cells transfected with RBP2 expression plasmid or cotransfected with miR-21 mimics and RBP2 expression plasmid, Cell proliferation assay and Soft agar assay of colony-formation suggested that RBP2 overexpression-mediated proliferation inhibition was partly relieved. It shows that miR-21 mediates the function of RBP2 at least in part. In addition, qRT-PCR showed that miR-21 was overexpressed in CML-BP compared to CML-CP.(7) PDCD4, which is a target gene of miR-21,promotes cell differentiation and inhibits cell proliferationIn K562 and HL60 cells transfected with miR-21 mimics/inhibitor, qRT-PCR and Western Blot suggested that PDCD4 was negatively regulated by miR-21.In differentiated K562 and HL60 cells induced by DMSO or ATRA, PDCD4 expression was increased. Cell proliferation assay and Soft agar assay of colony-formation showed that K562 and HL60 cells transfected with PDCD4 expression plasmid proliferated at a slower rate than with vector transfection, and their colony-formation ability was impaired.Conclusions:(1) RBP2 is underexpressed in CML-BP.(2) RBP2 induces leukemia cell differentiation and inhibits proliferation.(3) miR-21 is a key target gene of RBP2. By binding to miR-21 promoter region, RBP2 reduces H3K4 trimethylation and dimethylation at the proximal promoter region of miR-21 and inhibits miR-21 expression.(4) RBP2 induces cell differentiation and inhibits cell proliferation depending on miR-21, which negatively regulates PDCD4 expression.Part 2 MiR-370 sensitizes chronic myeloid leukemia K562 cells to homoharringtonine by targeting Forkhead box MlMicroRNAs (miRNAs) is a kind of endogenous non-coding RNAs, only 20-25 nucleotides. Binding to 3’untranslated region (3’UTR) of target genes, miRNAs inhibit gene expression post-transcriptionally or degrade mRNA. Deregulaton of miRNAs is related to many kinds of tumors and affects various biological effects, such as development, proliferation, differentiation and apoptosis, etc. Even more, recent studies suggest that miRNAs play an important role in drug resistance and drug sensitivity.Homoharringtonine (HHT), a traditional Chinese medicine, is a plant alkaloid separated from Cephalotaxus Plants. HHT has been successfully used in various leukemia treatments. HHT mainly kills cells in G1 and G2 phase, inhibits protein synthesis, induces cell differentiation and promotes cell apoptosis. In imatinib-resistant cell lines and CML-BP primary cells, HHT has synergistic activity with imatinib. Besides, HHT also cooperates with Ara-C and IFN-α. Phase I and II studies in the United States approved the clinical efficacy of HHT in CML but documented a high incidence of side-effect. Therefore, in order to reduce the dosage of HHT and decrease the incidence of side-effect, finding the new targets is the key point currently, which have cooperative actions with HHT.Purpose:In order to study the role of miR-370 in blast crisis of chronic myeloid leukemia and the relative mechanism, we determine miR-370 expression in CML-CP and CML-BP samples, study the effects of miR-370 on apoptosis and HHT-induced apoptosis.Methods:(1)Detect the expression of miR-370 in CML-CP and -BP samples by qRT-PCR.(2) Study the role of miR-370 in CML-BP cell line K562① Examine the effects of miR-370 on cell apoptosis and HHT-induced apoptosis by Flow CytometryK562 cells were treated with miR-370 mimics and/or HHT, divided into five groups: NC, miR-370 mimics, HHT, HHT+NC and HHT+miR-370 mimics. Detect cell apoptotic rate in these five groups.Transfecting miR-370 inhibitor into HHT-treated K562 cells, Flow Cytometry detects cell apoptotic rate of cells treated with HHT+NC and HHT+miR-370 inhibitor.② Detect the role of FoxM1 in miR-370-mediated apoptosis by Flow CytometryK562 cells were treated with miR-370 mimics and/or FoxM1 expression plasmid, including miR-370 mimics, FoxMl expression plasmid, and miR-370 mimics+FoxM1 expression plasmid. Detect cell apoptotic rate in these three groups.Transfecting miR-370 inhibitor and/or FoxM1 siRNA into K562 cells, Flow Cytometry detects cell apoptotic rate of cells treated with miR-370 inhibitor, FoxMl siRNA and miR-370 inhibitor+FoxM1 siRNA.(3) Study the regulation between HHT and miR-370, FoxMlAfter K562 cells were incubated with HHT for 72 hours at different concentrations, miR-370 and FoxMl expression were detected by qRT-PCR and Western Blot. In K562 cells incubated with HHT for 72 h and 96 h, the level of miR-370 and FoxMl were determined by qRT-PCR and Western Blot.Results:(1) Misregulation of miR-370 and FoxMl in CML-CP and CML-BP samplesIn 23 CML-CP samples,10 CML-BP samples and 14 healthy controls, qRT-PCR showed that compared to healthy controls, the level of miR-370 was lower in CML-CP samples, and lowest in CML-BP samples. Conversely, the level of FoxMl was higher and higher, accompany with the transition from CML-CP to CML-BP.(2) Ectopic expression of miR-370 sensitizes K562 cells to HHTK562 cells were treated with HHT or miR-370 mimics or HHT+miR-370 mimics. Flow Cytometry showed that miR-370 induced cell apoptosis, and it enhanced HHT-induced apoptosis.(3) Increased sensitivity to HHT with miR-370 overexpression was at least partially attributed to FoxM1 downregulation.K562 cells were treated with miR-370 mimics and/or FoxM1 expression plasmid, Flow Cytometry showed that FoxMl overexpression partly inhibited miR-370 overexpression-induced apoptosis. Conversely, In cells treated with miR-370 inhibitor and/or FoxM1 siRNA, Flow Cytometry showed that FoxMl siRNA partly promotes miR-370 underexpression-inhibited apoptosis.(4) HHT upregulates miR-370 expression in K562 cells.After K562 cells were incubated with HHT for 72 hours at different concentrations, qRT-PCR showed that miR-370 was upregulated in dose-dependent manner. On the other hand, in K562 cells treated with HHT for 72 h and 96 h, qRT-PCR showed that miR-370 was upregulated in time-dependent manner.Conclusions:(1) The level of miR-370 was lower in CML-CP, and lowest in CML-BP.(2) Ectopic expression of miR-370 sensitizes K562 cells to HHT.(3) Increased sensitivity to HHT with miR-370 overexpression was at least partially attributed to FoxM1 downregulation.(4) HHT upregulates miR-370 expression in time and dose-dependent manner.