Re-expression Of MiR-150Induces EBV-positive Burkitt Lymphoma Differentiation By Modulating C-Myb

BackgroundBurkitt lymphoma (BL) is a highly aggressive B-cell malignancy identified and described for the first time by Dennis Burkitt in1958. There are3major subgroups of BL that differ in geographic distribution and clinical manifestation:endemic, sporadic and immunodeficiency-associated. According the Epstein-Barr virus (EBV) infection status, BLs are divided into2subgroups:EBV-positive and EBV-negative BL. BLs have a tendency to morphologically resemble germinal center (GC) cells, and to immunophenotypically express characteristic GC cell markers such as CD10and BCL6, and coexpress B-cell markers CD19and CD20, which suggests follicle center B-cell origin for this lymphoma. In recent years, efforts have focused on improving therapy for this the fastest growing human tumor while minimizing treatment-associated toxicity. Outcome with intensive chemotherapy has improved and is now excellent in children, but the prognosis is poor in elderly adults. In addition, there are significant associated toxicities, such as frequent myelosuppression, mucositis, neuropathy, and complication of tumor lysis syndrome. Therefore, it is imperative and important to pursuit more effective therapies.MicroRNAs (miRNAs), a novel class of small noncoding RNAs of-19-26nt, exert multiple cellular functions and play critical roles in cellular proliferation, apoptosis, cellular differentiation and tumorigenesis. MiRNAs expression profiling studies have found that in hematopoiesis, certain miRNAs are expressed in a stage-specific fashion in the lymphoid hematopoietic system. Therefore, deregulations of their expression may result in the development of hematopoietic malignancies.MiR-150has attracted our great attention in hematopoiesis among a large number of miRNAs in recent years, which has been studied in B, T, and NK/T cells. There are strong evidences that miR-150is preferentially expressed in mature lymphocytes, but not their progenitors. Ectopic miR-150expression of murine hematopoietic stem cells impairs the transition of pro-B to pre-B stage. In addition, miR-150is found to target c-Myb, which is highly expressed in lymphocytes progenitors, down-regulated as maturation, and again increased after activation of the mature cells. It has been shown that expression of miR-150was consistently down-regulated in diffuse large B-cell lymphoma (DLBCL) cell lines compared with centroblasts. Furthermore, aberrantly low expression of miR150is identified in Sezary syndrome and NK/T-cell lymphoma. Re-expression of miR-150in NK/T-cell lymphoma cells increases the incidence of apoptosis and reduces cell proliferation, suggesting that miR-150functions as a tumor suppressor.The discovery of miRNAs has added an entirely new dimension to antitumor therapeutic approaches. Depending on miRNA function and status in tumor, miRNAs are generally classified as tumor suppressors or oncomiRs. Therefore, from the therapeutic point of view, tumor suppressor-miRNAs can be induced by ectopic expression, and those oncogenic-miRNAs can be inhibited. Re-expressing lost miRNA in a cell can deliver a dramatic effect, because miRNAs regulate a vast number of genes and pathways. The ectopic expression of miR-223, miR15/miR-16, let7, mir-342, miR-29a and miR-142-3p in acute myeloid leukemia could stimulate myeloid differentiation of leukemia cells. Therefore, differentiation induction by miRNAs is proposed to be a potentially attractive strategy for leukemia. Recently, our group found that CD99can trigger Hodgkin/Reed-Sternberg cells redifferentiation by upregulating miR-9-modulated PRDM1/BLIMP1. Here, we found that Burkitt lymphoma cell lines exhibited an extremely low expression of miR-150. Consequently, testing whether transfection of miR-150into BL cell lines can shift the regulators expression profiles toward that of the terminal B-cell is one purpose in our works. Although in immature hematopoietic system of mouse model, miR-150functions by targeting c-Myb, it is remain unknown whether there is the similar relationship miR-150with c-Myb in BL, which is another one question we’ll explore in our works.Objective1. Explosive expression profiles of miR-150and c-Myb in human B cell subpopulations from tonsils and in human peripheral lymphoma cell lines2. Constructing cell sublines Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150with stable expression of miR-1503. Explosive the possibility and potential mechanism that re-expression of miR-150induces Burkitt lymphomas differentiationMethod1. Expression profiles of miR-150and c-Myb in human B cell subpopulations from tonsils and in human peripheral lymphoma cell linesSelection of B cell subpopulations by FACS according the corresponding surface markers:B lymphocytes CD19+, naive cells CD19+CD38-IgD+, centroblasts CD19+CD38+IgD-, centrocytes CD19+CD38+IgD-, memory cells CD19+CD38-IgD-. Analysis of miR-150and c-Myb in B cell subpopulations from tonsils and in human peripheral lymphoma cell lines by real-time PCR. Analysis of c-Myb in human peripheral lymphoma cell lines by Western Blot. 2. Construction of cell sublines Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150stably expressing miR-150and analysis of their biological characteristicsStable expression lentiviral vectors for miR-150were provided by the Shanghai GeneChem (China), and the lentiviral supernatant was synthesized by GeneChem. Raji, Daudi, Ramos and BJAB were transfected by lentiviral supernatant (experimental group and control group), and the cell sublines were namely respectively Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150, all of which were tagged by GFP including control, and then sorted for GFP expression on a FACS aria. Transfection efficancy was confirmed by real-time PCR. The proliferation and apoptosis rate were examined by flow cytometry and MTT test in cell sublines.3. Re-expression of miR-150induces EBV-positive Burkitt lymphomas differentiationAnalysis of germinal center marker CD10, B lymphocyte marker CD19and plasma cell markers CD38and CD138in cell sublines by flow cytometry. Analysis of BCL6, PRDM1and bcl-2in cell sublines by real-time PCR, Western Blot and confocal.4. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomasAnalysis of c-Myb in cell sublines by real-time, Western Blot and confocal. Transient transfections with inhibition of miR-150in Daudi-miR-150and Raji-miR-150as well as c-Myb in Raji and Daudi cell lines were performed. Then BCL6, PRDM1and bcl-2were analyzed by real-time and Western Blot in c-Myb RANi Raji and Daudi cell lines. Analysis of c-Myb in miR-150inhibition Daudi-miR-150and Raji-miR-150cells by Western Blot. 5. Explosive of underlying mechanism by which miR-50was dysregulated in Burkitt lymphomasAnalysis of c-Myc in Burkitt lymphomas and cell sublines by real-time PCR and Western Blot. Analysis of miR-150in c-Myc RNAi Daudi cell line by real-time PCR.Statistical analysisAll images such as western Blot and flow cytomety are representative of at least three independent experiments. QRT-PCR assays were performed in triplicate for each experiment. The data shown are presented as the mean±standard derivation (SD) for three independent experiments. The SPSS software13.0was used for the statistical analysis. Differences are considered statistically significant at p<0.05, assessed using the one-way ANOVA for assays with qRT-PCR, and two-tailed independent Student’s t test for MTT assays and flow cytometry.Results1. Expression profiles of miR-150and c-Myb in human B cell subpopulations from tonsils and in human peripheral lymphoma cell lines(1). Selection of B cell subpopulations by FACSFive B cell subpopulations were selected according the surface markers:B lymphocytes CD19+, naive cells CD19+CD38-IgD+, centroblasts CD19+CD38+IgD-, centrocytes CD19+CD38+IgD-, memory cells CD19+CD38-IgD-.(2). Expression profiles of miR-150and c-Myb in B cell subpopulations from tonsilsWe employed qRT-PCR to analyze expression pattern of miR-150in these four B cell subsets. The result showed that the expression pattern of miR-150of four B cell subpopulations was dynamic. Additionally, we determined the expression levels of c-Myb in the four B cell subsets. A reciprocal correlation of expression pattern of miR-150and c-Myb was found. (3). Expression profiles of miR-150and c-Myb in human peripheral lymphoma cell linesWe detected the levels of expression of miR-150in human peripheral lymphoma cell lines i.e. Daudi, Raji, Ramos, BJAB, KM3, L428, KARPAS-299and Jurkat. The result showed that miR-150expression was significantly reduced in all cell lines with respect to normal B cell. What’s more, we analyzed the expression of c-Myb in lymphoma cell lines in the mRNA levels and in the protein levels. The results demonstrated that the most of these lymphoma cells strongly expressed c-Myb.2. Construction of cell sublines Raji-miR-150, Daudi-miR-150, Ramos-miR-150and BJAB-miR-150stably expressing miR-150and analysis of their biological characteristics(1).The design and synthesize of lentiviral vector for miR-150Lentiviral vector for miR-150(PP-GFP) and control lentiviral vector in this experiment were used, both of which were provided by the Shanghai GeneChem (China).(2).Construction of cell sublines with stable expression of miR-150Lentiviral vectors constitutively expressing pre-miR-150labeled with GFP were stably transfected into Daudi, Raji, Ramos and BJAB along with a control vector labeled with GFP. GFP+cells were selected by FACS. The efficiency of transfection was measured by qRT-PCR. The result showed that transfected cells expressed activity of miR-150in the vicinity of physiological levels.(3).Examination of proliferation and apoptosis in cell sublinesWe monitored weekly the GFP expression in the mixed cultures with GFP-miR-150positive (GFP-miR-150+) cells and GFP negative (GFP-) cells by flow cytometer. Our results showed that there was a significant reduction in%GFP-miR-150+compared with GFP-in Daudi-miR-150and Raji-miR-150but not in BJAB-miR-150and Ramos-miR-150, then, the results were confirmed by MTT test. We found that re-expression of miR-150resulted in a strong increase in the early apoptotic fraction and late apoptotic cells in Daudi-miR-150but not in Raji-miR-150, Ramos-miR-150and BJAB with respect to GFP+control.3. Re-expression of miR-150induces EBV-positive Burkitt lymphoma differentiation(1). Analysis of BCL6, PRDM1and bcl-2in cell sublinesOur results showed that re-expression of miR-150resulted in a consistent down-regulation of BCL6in the protein and mRNA levels compared with controls in Daudi-miR-150, Raji-miR-150. On the other hand, re-expression of miR-150induced PRDM1and bcl-2expression in the protein and mRNA levels in Daudi-miR-150and Raji-miR-150. The results were further confirmed by confocal analysis in Raji-miR-150. No obvious alterations were observed in Ramos-miR-150and BJAB-miR-150about expression of BCL6, PRDM1and bcl-2compared with controls.(2). Analysis of CD10, CD19, CD38and CD138in cell sublinesOur results indicated that there was a significant increase in the percentage of CD19-CD10-CD138+cells in Daudi-miR-150, and CD10-CD38++cells in Raji-miR-150compared with empty vectors (GFP). No changes were observed in Ramos-miR-150and BJAB-miR-150about immunophenotypic profiles compared with GFP.(3).Test of cell size and intracellular organelles in cell sublinesBy analyzing SS and FS, the optical physical parameters of flow cytometry, we found that intracellular organelles were significantly increased in both Daudi-miR-150and Raji-miR-150compared with GFP. Furthermore, Raji-miR-150displayed increased cellular size. 4. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas(1). Analysis of c-Myb in cell sublinesThe results demonstrated that although c-Myb protein was down-regulated in Daudi-miR-150and Raji-miR-150cells compared with controls, there was different expression profiling in these cells in the mRNA levels in comparison to controls:no obvious alteration and significant downregulation, respectively. The confocal analysis also displayed that c-Myb protein was reduced in Raji-miR-150compared with GFP. The miR-150-mediated effect on c-Myb in Ramos-miR-150and BJAB-miR-150could not be found.(2). Analysis of c-Myb in miR-150inhibition Daudi-miR-150and Raji-miR-150cellsThe results displayed that the expression of c-Myb protein increased again compared with controls when miR-150expression was inhibited in Daudi-miR-150and Raji-miR-150, determined by Western Blot.(3).Analysis of BCL6, PRDM1and bcl-2in c-Myb RNAi Raji and Daudi cell linesThe c-Myb siRNA Raji and Daudi cells displayed downregulation of BCL6and upregulation of bcl-2in the levels of mRNA and protein compared with controls. While PRDM1mRNA was induced in c-Myb siRNA Raji and Daudi cells, Western blot analysis showed that PRMD1protein levels only were detectable in c-Myb siRNA Daudi cells, and flow cytometry analysis showed that there were no changes of phenotypes in c-Myb siRNA Raji and Daudi cells compared with controls.5. Explosive of underlying mechanism by which miR-50was dysregulated in Burkitt lymphomas(1). Analysis of c-Myc in Burkitt lymphomas and cell sublines The results revealed that they strongly expressed c-Myc protein. Additionally, re-expression of miR-150led to downregulation of c-Myc protein in Daudi-miR-150and Raji-miR-150compared to controls, whereas had little effect on c-Myc transcripts.(2). Analysis of miR-150in c-Myc RNAi Daudi cell lineWe knocked down expression of c-Myc in Daudi cell line. The efficacy of knockdown was examined by real-time PCR. The result showed that miR-150was induced in low-status c-Myc.Conclusion1. The expression pattern of miR-150and c-Myb in human four B cell subpopulations was dynamic. And a reciprocal correlation of expression pattern of miR-150and c-Myb was found, which also can be observed in human peripheral lymphoma cells, that is, miR-150was lowly expressed or absent and c-Myb was highly expressed.2. Re-expression of miR-150has different influence on biological characteristics in EBV-positive and EBV-negative Burkitt lymphomas.3. Re-expression of miR-150can induce EBV-positive Burkitt lymphomas Raji and Daudi differentiation toward plasma cell, but no effect on EBV-negative Burkitt lymphoma Ramos and BJAB.4. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas.5. Dysregulated c-Myc contributes to repressed miR-150.Innovation points1. The dynamic expression pattern of miR-150and c-Myb in human four B cell subpopulations.2. Re-expression of miR-150has different influence in EBV-positive and EBV-negative Burkitt lymphomas. 3. C-Myb is a potential target of miR-150in EBV-positive Burkitt lymphomas.