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The Mechanisms of CD99 Regulation on miR-9/PRDM1 Inducing Redifferentiation of H/RS Cells

Author: HuangXuePing
Tutor: ZhaoTong
School: Southern Medical University,
Course: Pathology and Pathophysiology
Keywords: Classical Hodgkin lymphoma CD99 miR-9 PRDM1/BLIMP1 Redifferentiation
CLC: R733.1
Type: Master's thesis
Year: 2011
Downloads: 52
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Abstract

Background studies have shown that the occurrence of malignant tumors and cell differentiation disorder or differentiation block is closely related to cancer treatment regardless of radiotherapy, chemotherapy or molecular targeted therapy and biological therapy, therapeutic concepts are taken to kill tumor cells, these treatment method to kill tumor cells while killing effect on normal cells. A growing number of studies have shown that induced to differentiate in vitro and in vivo agent, the many signs of the malignant cell morphology, biology or biochemistry evolution of differentiation to the direction of the normal cells, even totally normal cells into or near normal cells, a phenomenon known as the re-differentiation of the tumor cells (redifferentiation), thereby inducing the differentiation therapy of cancer therapy will be the future of a new development direction. Hodgkin's lymphoma (Hodgkin lymphoma, HL) is a lymphoid malignancies of the hematopoietic system, the earliest described in 1832 by Thomas Hodgkin in the UK, including classical Hodgkin lymphoma (classic Hodgkin lymphoma, cHL) accounting Hodge lymphoma 95% of the cases. The typical form of the disease is characterized by a small number of neoplastic H / RS (with Hodgkin '/ Reed-sternberg) cells (lt; 1%) infiltration in the inflammatory reaction background cells. In more than 170 years, the nature of the H / RS cells as well as the development of mechanisms has been plagued by the medical profession. Kuppers microdissection from tissue sections picked up a single H / RS cells by single-cell PCR technology analysis Ig gene rearrangement confirm the most of the H / RS cells from lymphoid organs germinal center (germinal center, GC) -apoptotic B cells, also called \Looking for H / RS cells during differentiation block key target, adjust the \Reported antisense CD99 expression plasmid transfected human B lymphoma cell line BJAB and IM9, morphology and phenotype H / RS cells are very similar, suggesting that CD99 expression lowered H / RS cells form a key molecular events. Transcription factor PRDM1, induction of differentiation of B cells to plasma cells of a switch, is a crucial regulatory factor required for plasma cell differentiation. Studies suggest that PRDM1 is a tumor suppressor gene in cHL functional inactivation of blocking the differentiation of B cells to mature plasma cells may cHL pathogenic important reason. H / RS cells with early plasma cell differentiation potential, induced by H / RS cells to the direction of the end of B cell differentiation checkpoints must open PRDM1 plasma cell regulation. Recent studies have found that miRNA play an important role in the differentiation of B cells and B-cell malignancies. Nie luciferase reporter experiments confirmed the direct target of miR-9 regulation in cHL cell lines L428 PRDM1, miR-9 can negatively regulate PRDM1 expression. Therefore, miR-9 may be another important target for the H / RS cell differentiation arrest. Our group pre-built cHL cell lines L428 cell sublines stable over-expression of the CD99 gene, named L428-those of CD99 by immunocytochemistry in this study on the basis of real-time quantitative RT-PCR, Western blot, immune confocal situ hybridization, miRNA antisense oligonucleotide instantaneous transfection, bioinformatics analysis, after the in L428-CD99 cell subline series identification, observation raised the L428 cells after CD99 B cell differentiation table type, cell morphology and cell biology behavior change explicitly raised the CD99 H / RS cells can reproduce the B-cell phenotype, open to the plasma cell differentiation switch gene PRDM1 induction to the direction of the end of B cell differentiation possibility and regulatory mechanism to verify the possibility of miR-9 expression in IH / RS cells, target PRDM1 regulation of relations and explore induced differentiation, and provide a reference for the development of mechanisms for further elaboration of the H / RS cells. Purpose This study intends through four parts: those of CD99 and PRDM1 expression in of cHL tissue and cellular level observations CD99 and PRDM1 lymph node reactive hyperplasia (\difference in the expression of cell lymphoid hematopoietic tumor cell lines. Those of CD99 regulation of the PRDM1 the expression induced by H / RS cell differentiation to explore CD99 and PRDM1 regulation relationship explicitly raised the CD99 gene diagnostic marker of cHL cell lines L428 cells, B-cell phenotype, and plasma cells phenotype reveals CD99 The gene's role in cHL and differentiation. , MiR-9 expression in H / RS cells and quantitative targets PRDM1 regulation and positioning transfection silence validate miR-9 expression in L428 cells and target PRDM1 regulation. , CD99 and miR-the mutual regulation preliminary study of the relationship between the preliminary experiment by bioinformatics analysis of CD99 and miR-9 mutual regulatory network and explore those of CD99 regulation of miR-9 and PRDM1 relationship. Research content and methods, CD99 and PRDM1 in the level of expression of cHL tissues and cells collected cHL and RH tissue, through the CD99 and PRDM1 the immunohistochemistry compare expression differences in the organization, and further by Western blot and immunocytochemistry Methods to detect the expression of CD99 and PRDM1 in six of B-cell origin and three T-cell lymphoma cell lines. Second, those of CD99 the regulation PRDMl the expression induced by H / RS cell differentiation 1.L428-CD99 cell subline identification of pre-build lentiviral plasmid stability over L428-CD99 expression of CD99 gene clone carrier green fluorescence was observed by fluorescence microscopy protein expression using real-time quantitative RT-PCR, Western blot and immunofluorescence confocal detection of CD99 mRNA and protein expression. 2.CD99 overexpression morphological and biological characteristics of cHL cell lines L428 MTT assay HE staining and phalloidin staining CD99 overexpression L428 cell proliferation, shape and size, and cytoskeletal proteins . 3.CD99 upregulated on differentiation-related protein in L428 cells by Western blot and immunofluorescence confocal detection of the PRDM1 protein before and after the over-expression of the CD99 gene expression; using immunocytochemistry and flow cytometry cHL diagnostic markers (CD30 and CD15) B cell differentiation antigen (CD10, CD19, CD20, CD45, CD79α, BCL6, PAX5 and MUM1), and plasma cell markers (CD38, neither CD138 nor). Third, miR-9 expression in H / RS cells and target PRDM1 regulation. Detection of miR-9 expression in L428 cells using immunomagnetic beads sorting normal lymph node CD19 B lymphocytes as control; real-time fluorescence quantitative RT-PCR and in situ hybridization detection of CD19 B lymphocytes and 8 L428 cell lines, including lymphoma cell lines miR-9 expression. Instantaneous interference miR-9 L428 cells PRDM1 protein expression and proliferative capacity miR-9 antisense oligonucleotides transiently transfected L428 cells, fluorescent microscopy analysis transfection efficiency, real-time quantitative RT-PCR assay interference efficiency ; Western blot and immunofluorescence confocal detection knock PRDM1 protein expression the low of miR-9; MTT assay miR-9 antisense oligonucleotide L428 cell proliferative capacity. Those of CD99 and miR-9 mutual regulation of the relationship between the preliminary study using real-time quantitative RT-PCR analysis before and after CD99 overexpression of miR-9 expression; using real-time quantitative RT-PCR and Western blot detection before and after miR-9 interference CD99 expression; signal regulatory pathway between CD99 and miR-9 through bioinformatic analysis. Results, CD99 and PRDM1 expression in cHL tissue and cellular level. Organization RH, CD99 expressed mainly in the the lymphoid follicles jacket District and follicular area of ??PRDM1 Ming with germinal centers were scattered in the weak expression; 62 cases cHL organization, only one case of CD99 positive expression in H / RS cells in all cases the PRDM1 showed a negative expression. 2.CD99 and PRDM1 protein in the multiple plasma cell myeloma cell line RPMI-8226 express higher expression in L428 cells and other B-cell origin of lymphoma cell lines lower or absent. Those of CD99 regulation of the PRDM1 the expression induced by H / RS cell differentiation 1.L428-CD99 cell subline are visible under a fluorescence microscope strong green fluorescence real-time quantitative RT-PCR, Western blot and confocal microscopy show The bare cell group and the empty vector group of, L428-CD99 the cell subline CD99 gene and protein expression were significantly increased. 2.MTT detected those of CD99 gene over-expression group than in the empty vector proliferation slows down, the difference was significant (F = 305.917, P = 0.000); HE staining count L428-those of CD99 cell subline 20 uM cells to total the ratio of the number of cells (50.187 ± 2.518)%, compared with the control group of cells (77.588 ± 5.580)% low proportion, the difference was significant (Z = -2.611, P = 0.008); ghost than cyclic peptide staining with the control group. than, L428-CD99 cell filopodia disappeared, the thinning of the cortical actin. Western blot and confocal detection results show that, relative to the group and empty vector group of bare cells, CD99 gene overexpression the group PRDMl protein expression increased significantly; immunocytochemistry and flow cytometry found that those of CD99 gene overexpression, and control group, CD30, CD15, and MUM1 reduced expression of CD10, CD19, CD79αBCL-6 increased of PAX5 and CD38 expression, CD20 and CD138 expression did not change significantly. Three miR-9 in the expression of the H / RS cells and target PRDM1 regulation real-time quantitative RT-PCR analysis found that compared to sorting CD19 B cells and other cell lines, miR-9 in L428 cells of higher expression, the differences were statistically significant (P lt; 0.05); situ hybridization showed that the expression of miR-9 is located in the cytoplasm in L428 cells showed diffuse strong positive expression in DLBCL and Burkitt's lymphoma cell lines were scattered weakly positive expression, negative expression in T-cell origin] KARPAS-299 and Jurkat cells. 2 real-time quantitative RT-PCR analysis found that, compared with the control group, miR-9 expression levels of miR-9 antisense oligonucleotide group dropped about 50%, the difference was significant (t = -5.208, P = 0.035). 3. Western blot and immunofluorescence confocal results show that miR-9 antisense oligonucleotide group PRDM1 protein expression relative to the bare cell group and the control group was significantly higher. 4.MTT detected the bare cell group and the control group (NCi) and miR-9 antisense oligonucleotide transfection group (miR-9i) cell proliferation activity differences between different groups was not statistically significant (F = 1.069 , P = 0.350). Fourth, CD99, and miR-9 mutual regulation of the preliminary study of the relationship between a real-time quantitative RT-PCR results show that, CD99 transfected L428 cells group relative to the empty vector expression of miR-9 levels were significantly lower, the difference was statistically significant ( t = 282.071, P = 0.000). Real-time fluorescence quantitative RT-PCR detection discovery of miR-9i group CD99's expression level and the control group (NCi) and bare cell group fairly, the difference does not have a significant sex (P = 0.719 and P = 1.000); Western blot results show with the control group (NCi) and bare cells, CD99 protein levels of miR-9 antisense oligonucleotide transfection group did not change significantly. 3. Bioinformatic analysis results show that the, CD99 may be connected through KPNB1 middle of SMAD3 and SMAD4 further and MYC role, regulation of miR-9 expression. Conclusion 1.CD99 and PRDM1 in of cHL tissue and cell lines was low or absent expression. 2.L428-CD99 cell subline overexpression carrier stability, the higher the level of those of CD99 gene and protein expression, cell proliferation slows down, smaller size, cytoskeleton remodeling; those of CD99 gene overexpression of H / RS cells lost cHL diagnostic markers reproduce the B-cell phenotype, raised the plasma cell differentiation key factor PRDM1 protein expression, a plasma cell labeling before plasmablastic differentiation characteristics. 3.miR-9 in L428 cell specific high expression; miR-9 can negatively regulate PRDM1 expression. 4.CD99 can negatively regulate the expression of miR-9, miR-9 can not react on those of CD99; those of CD99 is likely through the regulation of miR-9 thereby regulating PRDM1 expression. Innovations. Arrest and differentiation of disorders of the H / RS cell generation and differentiation of B cells is closely related to those of CD99 revealed rniR-9 and of PRDM1 with cHL the development of closely related; revealed CD99 gene may regulate miR-9 mediated PRDM1 expression, to induce H / RS cells reproduce B-cell phenotype, and direction to the end of the B cell differentiation, providing help to elucidate the mechanism of cHL occurrence, and provide a theoretical basis for differentiation therapy and precise target with original innovation.

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