Hsa-miR-150 Induces B Cell-derived Lymphoma Cell Lines Differentiation Toward Terminal B Cell By Regulating C-myb

BackgroundIncidence of the lymphoma that is a primary malignant tumor of the lymphatic hematopoietic tissue, with the continuing deterioration of human living environment and increasing work pressure, has increased year by year. MicroRNAs (MiRNAs) are small, endogenous non-coding RNAs (about 22 nucleotides long), commonly found in plants and animals. So far, there were about 2,000 kinds of miRNAs were discovered. They were involved in cell proliferation, differentiation, apoptosis, embryo development and a series of important life processes. A recent study showed that many annotated human miRNAs located in the areas of the genome, known as fragile sites (frequent deletion or mutation in tumor cells), that were associated with cancer. Many miRNAs expression level is down-regulated, but a small number of miRNAs expression level is up-regulated in tumor tissues. This indicates that a lot of miRNAs might play the function role of oncogene or tumor suppressor gene.More and more evidences show that the tumor pathogenesis relates to disturbance of normal cell differentiation or differentiation arrest. The differentiation of tumor cells is a multi-gene disorders, multi-step process to change a single gene is difficult to have significant effect on cell differentiation. However, miRNAs can control hundreds of target genes, parts of which are transcription factors, and transcription factors themselves play a regulatory role. Therefore, changing the expression of a microRNA can exert a dramatic effect on cell differentiation. So induced differentiation therapy will be a new direction of development in the field of cancer treatment in the future. Induced differentiation therapy, known as the redifferentiation of tumor cells, refers to the effects under inducing agents, tumor cells are induced differentiation toward normal cells by changing tumor cells proliferation, invasion, metastasis and other malignant characteristics. In view of its prominent role in regulating cell differentiation，miRNAs may be a new target for induced differentiation therapy.Tumors of haematopoietic and lymphoid tissues, especially B cell lymphomas are an excellent model for induced differentiation therapy research. In domestic and international research at present, B lymphocyte differentiation spectrum is more indepthly studied compared to other hematopoietic cells. Different differentiation stage of B lymphocyte is in line with the corresponding lymphomas type with similar immunophenotypes. B lymphocyte lymphoma provides a very good research platform to study miRNAs induced tumor cell differentiation.Mature hsa-miR-150 locates 19q13.33 is composed of 22 nucleotide. It is highly expressed in haematopoietic and lymphoid tissues, while lowly expressed in else tissues. Expression level of hsa-miR-150 is gradually up-regulated with the gradual maturation of B lymphocyte development. However, its target gene c-myb expression level is exactly the opposite. Normal expression level of hsa-miR-150 is very critical for B lymphocyte differentiation, but its expression level in mature B cell lymphoma is not clear. If disordered expression of hsa-miR-150 was corrected, it is still not clear whether hsa-miR-150 could induce B cell-derived lymphoma cell lines differentiation toward terminal B cell. It is the critical problem that we will resolve. Objective1. Detecting expression level of hsa-miR-150 and c-myb in normal mature B lymphocyte and B cell lymphomas.2. Constructing two cell sublines (OCI-Ly10-miR-150 and Raji-miR-150) with stable expression of hsa-miR-150.3. Discovering mechanism of hsa-miR-150 induced B cell-derived lymphoma cell lines differentiation toward terminal B cell.Method1. Expression level difference of hsa-miR-150 and c-myb between normal B cells and lymphoma cell lines.Identifying biological function and immunophenotype of three diffuse large B cell lymphoma (DLBCL) cell lines by using HE stain, immunocytochemistry, and MTT technology; Detecting expression level of hsa-miR-150 in normal mature B lymphocyte and B cell lymphoma by using real-time PCR and in situ hybridization technology; Detecting expression level of c-myb from the perspective of quantitative and positioning respectively by using western blotting and immunofluorescence cytochemistry technology.2. Construction of B lymphoma cell sublines (Raji-miR-150 and OCI-Ly10-miR-150).First, lentiviral expression vector included objective gene is extracted. Secondly, E. coli liquid of lentiviral expression vector included objective gene was sent to invitrogen sequencing. DNA sequences and the flanking sequences of hsa-miR-150 with corresponding sequences published in GenBank are consistent by blast alignment analysis. Thirdly, envelope plasmid (pMD2.G), packaging structure of plasmid (psPAX2) and lentiviral expression vector included objective gene were co-transfected to 293FT cell lines by LipofectamineTM 2000 and viral supernatant was collected after transfection. Fourthly, lentiviral supernatant (experimental group and control group) containing recombinant plasmids were transfected to Raji and OCI-Ly10 cell lines. To identify biological function of two cell sublines by using MTT, cell cycles and apoptotic rate detection by FACS.3. hsa-miR-150 induces B cell-derived lymphoma cell lines differentiation toward terminal B cell by regulating c-myb.To detect expression levels of Pax5、Bcl-6、IRF4、PRDM1 and XBP-1 in mRNA level in OCI-Ly10, OCI-Ly10-miR-150, Raji and Raji-miR-150 cell lines by real-time PCR. To detect expression levels of c-myb in mRNA and protein levels in OCI-Ly10, OCI-Ly10-miR-150, Raji and Raji-miR-150 cell lines by using real-time PCR and western blotting technology. To detect expression levels of Bcl-6 and PRDM1 in protein levels in OCI-Ly10, OCI-Ly10-miR-150, Raji and Raji-miR-150 cell lines by using western blotting.4. Statistical analysisThe experimental results were processed and analyzed by SPSS 13.0 statistical software. Measurement data was represented by mean±standard deviation (x±s) Different groups of cell proliferation ability by MTT assay was analyzed by the use of factorial design analysis of variance and single factor analysis of variance (one-way ANOVA), the difference among each groups was analyzed by LSD multiple comparisons. Expression level of hsa-miR-150 by real-time PCR was analyzed by the use of K independent samples test and 2 independent samples test. Detection of cell cycle and apoptotic rate by FACS was analyzed by the use of independent samples T test. Expression level of Pax5、Bcl-6、IRF4、PRDM1、XBP-1 and c-myb by real-time PCR was analyzed by the use of 2 independent samples test.Result1. Expression level difference of hsa-miR-150 and c-myb between normal B cells and lymphoma cell lines.(1) Identification of morphology, immunophenotype and biological function of three DLBCL cell linesOCI-Ly1 and OCI-Ly8 cell lines were centroblastic variants, and OCI-Ly10 cell line was immunoblastic variant. OCI-Ly1 and OCI-Ly8 cell lines were germinal center B cell-like (GCB) type and OCI-Ly10 cell line was activated B cell-like (ABC) or Non-GCB type. Cell proliferation ability detected by MTT was analyzed by the factorial design analysis of variance. There were significant difference among the different cell groups (F=34.321, P<0.001), LSD multiple comparison showed that the proliferation activity of OCI-Ly10 was significantly higher than that of OCI-Ly1 and OCI-Ly8 (P<0.001), however, there was no statistical difference between the proliferation activity of OCI-Ly1 and OCI-Ly8.(2) Expression level difference of hsa-miR-150 between normal B lymphocytes and lymphoma cell lines.Expression level of hsa-miR-150 by real-time PCR was analyzed by K independent samples test and 2 independent samples test. Expression level of hsa-miR-150 in CD19+ B cells was significantly higher than that of the different lymphoma cell lines (P≤0.05). Expression level of hsa-miR-150 was very weak or negative in OCI-Ly1, OCI-Ly8, OCI-Ly10 and Raji cell lines by in situ hybridization.(3) Expression level of c-myb in lymphoma cell lines.Expression level of c-myb (72KDa) was detected from the perspective of quantitative by the western blotting in the protein level. Expression level of c-myb in Karpas299, Jurkat, Raji and OCI-Ly10 cell lines was higher than that in OCI-Ly1 and OCI-Ly8 cell lines. Expression level of c-myb in L428 cell line was negative. And expression level of c-myb (72KDa) was detected from the perspective of positioning by immunofluorescence cytochemistry under co-focal fluorescence microscope. Expression level of c-myb in Jurkat, Raji and OCI-Ly10 cell lines was strong, however, weak in OCI-Ly8 cell line.2. Constructing two cell sublines (OCI-Ly10-miR-150 and Raji-miR-150) with stable the expression of hsa-miR-150.(1) Sequencing of the recombinant lentiviral vector (pEZX-miR-150-eGFP)DNA sequences and the flanking sequences of hsa-miR-150 by the invitrogen sequencing with corresponding sequences published in GenBank are fully consistent by blast alignment analysis.(2) Packaging of the recombinant lentiviral vector (pEZX-miR-150-eGFP)Envelope plasmid (pMD2.G), packaging structure of plasmid (psPAX2) and lentiviral expression vector included objective gene were co-transfected to 293FT cell lines by LipofectamineTM 2000. A great deal of green fluorescence was observed under fluorescence microscope. It was confirmed that abundant 293FT cells were transfected with plasmids and the transfection efficiency was about 90%.(3) Raji and OCI-Ly10 were transfected by lentiviral supernatant containing recombinant plasmids.Raji and OCI-Ly10 cell lines were transduced with the lentiviral supernatant containing recombinant plasmids. A great deal of green fluorescence was seen under fluorescence microscope. It was confirmed that plasmids containing recombinant were transferred into Raji and OCI-Ly10 cells, and the transfection efficiency was about 80%-90%. Expression level of hsa-miR-150 was analyzed by 2 independent samples test. Expression level of hsa-miR-150 after transfection was 595 and 44 times compared to the before transfection in Raji and OCI-Ly10 cells respectively (Wilcoxon W=6.000, P=0.037)(4) Comparison of the cell proliferation ability by MTT before and after transfection of hsa-miR-150 hsa-miR-150 in Raji cell. The cell proliferation ability detected by MTT was analyzed by the factorial design analysis of variance. The proliferation activity of Raji-control was significantly higher than that of Raji-miR-150 cells (F=171.451, P<0.001)(5) Comparing the cell cycle by FACS before and after transfection of hsa-miR-150 in Raji cell.The cell proliferation rate (S+G2 %) detected by FACS was analyzed by the independent sample T test. The cell proliferation rate of Raji-control was significantly higher than that of Raji-miR-150 cells (t=23.855, P=0.002)(6) Comparison of the apoptotic rate by FACS before and after the transfected hsa-miR-150 in Raji cell.The cell apoptotic rate detected by FACS was analyzed by the independent sample T test. The cell apoptotic rate of Raji-miR-150 was significantly higher than that of Raji-control cells (t=-18.651, P<0.001)3. hsa-miR-150 induces B cell-derived lymphoma cell lines differentiation toward terminal B cell by regulating c-myb.(1) Comparison of expression level of immunophenotype related B lymphocyte differentiation before and after transfection hsa-miR-150.Expression level of immunophenotype related B lymphocyte differentiation by real-time PCR was analyzed by 2 independent samples test. Expression levels of Pax5 and Bcl-6 in Raji-miR-150 cells were lower than that of Raji-control cells (Wilcoxon W=6.000, P=0.037). Expression levels of IRF4, PRDMland XBP-1 in Raji-miR-150 cells were higher than that of Raji-control cells (Wilcoxon W=6.000, P=0.037)(2) Comparison of expression level of c-myb before and after transfection of hsa-miR-150 in Raji and OCI-Ly10 cells.Expression level of c-myb by real-time PCR was analyzed by 2 independent samples test. Expression level of c-myb in mRNA level in OCI-Ly10-miR-150 cell was significantly lower than that in OCI-Ly10-control cell (Wilcoxon W=6.000, P=0.037), and expression level of c-myb in mRNA level in Raji-miR-150 cell was statistical difference than that in Raji-control cell (Wilcoxon W=6.000, P=0.037). And expression level of c-myb in protein level was detected from the perspective of quantitative and positioning respectively by western blotting and immunofluorescence cytochemistry technology. Not only Expression level of c-myb in OCI-Ly10-miR-150 cell was significantly lower than that in OCI-Ly10-control cell, but also Expression level of c-myb in Raji-miR-150 cell was significantly lower than that in Raji-control cell.(3) Comparison of expression level of Bcl-6 and PRDM1 before and after transfection hsa-miR-150 in protein level in Raji and OCI-Ly10 cells.Expression level of Bcl-6 in protein level in both OCI-Ly10-miR-150 and Raji-miR-150 cells by western blotting was significantly lower than that in OCI-Ly10-control and Raji-control cells. As for the expression of PRDM1, there was no difference between Raji-miR-150 and Raji-control cells, but it was significantly higher in OCI-Ly10-miR-150 cells than that in OCI-Ly10-control.Conclusion1. Expression patterns of hsa-miR-150 and c-myb were opposite and hsa-miR-150 may down-regulate expression level of c-myb.2. Cell sublines (Raji-miR-150 and OCI-Ly10-miR-150) with stable expression of hsa-miR-150 were constructed. hsa-miR-150 can inhibit tumor cells proliferation, prompt tumor cells apoptosis.3. hsa-miR-150 induces B cell-derived lymphoma cell lines differentiation toward terminal B cell and the mechanism of induced differentiation is related to down-regulation of c-myb. Innovation points1. Expression level difference of hsa-miR-150 between normal B cells and lymphoma cell lines is not reported so far in the literature.2. To construct the cell sublines (Raji-miR-150 and OCI-Ly10- miR-150) with stable expression of hsa-miR-150 and our research result confirmed that hsa-miR-150 can inhibit tumor cells proliferation, promoting tumor cells apoptosis.3. hsa-miR-150 induces B cell-derived lymphoma cell lines differentiation toward terminal B cell. The mechanism of induced difference is related to down-regulation of c-myb.