Study Of Metadherin And Wnt/Beta-Catenin Signaling Pathway In Diffuse Large B-Cell Lymphoma

Diffuse large-B-cell lymphoma (DLBCL) is the most common type of lymphoma in adults, accounting for30%to40%of adult non-Hodgkin lymphoma (NHL). DLBCL is an aggressive disease with variable histomorphological, biological and cytogenetic features, as well as clinical outcomes. In the past few years, the introduction of antilymphoma monoclonal antibodies, notably rituximab, in combination with chemotherapy has significantly improved survival outcomes of patients with DLBCL. Nevertheless it is still recrudescent, drug-resistant and even fatal in a part of patients. Most lymphoma probably activates more than one survival signaling pathway and its malignization is a complex process with multifactor; therefore, clinical studies should be promoted to further investigate specific biomarkers and cellular signaling pathways, understanding molecular pathogenesis of DLBCL and developing more targeted and effective treatments.Metadherin (MTDH, also known as astrocyte elevated gene-1/AEG-1, and Lyric) was first cloned as an HIV-1, viral glycoprotein gp120or tumor necrosis factor a (TNF-α)-inducible gene in primary human fetal astrocytes (PHFAs). Human MTDH gene is located in8q22.1and consists of12exons and11intrones. MTDH mRNA is7667bp in length, and it encodes a582-amino acid protein with a predicted molecular mass of64kDa and isoelectric point of9.33.In recent years, MTDH, involved in aberrant proliferation, survival, and increased migration, invasiveness, and metastasis of tumor cells, has been demonstrated as a potentially crucial mediator of various types of human malignancies. Expression analysis revealed that MTDH expression is significantly higher in melanoma, breast, esophageal, gastric, hepatocellular, endometrial and prostate cancers, renal cell carcinoma, neuroblasoma, malignant glioma and osteosarcoma cell lines and tumor samples of patients compared with their normal counterparts. MTDH upregulation can promote survival, invasion and metastasis of tumor cells, and silence of MTDH can significantly inhibit clone formation and invasion and enhance cell apoptosis. MTDH promotes tumor progression by modulating multiple oncogenic signaling pathways, such as NF-κB, PI3K/Akt and Wnt/β-catenin pathways.Wnt/β-catenin signaling pathway acts a significant part in embryonic development and in maintenance of organs and tissues in adults. A lot of studies indicate that Wnt/β-catenin pathway involves in the pathogenesis of a range of disease including many kinds of carcinomas. Wnt/β-catenin pathway plays an important role in progression of several subtypes of lymphoma such as Epstein-Barr Virus (EBV)-positive Burkitt's lymphoma (BL), mantle cell lymphoma (MCL), cutaneous lymphoma, extranodal marginal zone lymphoma, DLBCL, among others. Therefore, further study on the status and regulation of Wnt/β-catenin signaling pathway in DLBCL will help to understand the pathogenesis of DLBCL.More recently, MTDH has been found to connect with Wnt/p-catenin pathway in hepatocellular carcinoma cells through the activation of the Raf/MEK/MAPK branch of Ras signaling pathway, leading to β-catenin nuclear translocation and upregulation of different target gene expressions. Inhibition of MTDH expression by specific siRNA clearly decreased the level of β-catenin and it may play a role in Wnt/β-catenin-mediated gastric cancer progressionMTDH and Wnt/β-catenin pathway are both involved in the pathogenesis of carcinomas, and they have connection with each other in hepatocellular and gastric carcinomas. However, the expression of MTDH in DLBCL and the relationship between MTDH and Wnt/β-catenin pathway in the pathogenesis of DLBCL have yet not been reported. For the first time, our study focuses on illuminating the role of MTDH and the relationship between MTDH and Wnt/β-catenin pathway in the pathogenesis of DLBCL by using cellular biology, molecular biology, immunology and gene silencing technologies. This novel study may contribute to further investigation on the useful biomarkers and potential therapeutic target in the DLBCL patients.Part I Screening and analysis of differentially expressed genes in diffuse large B-cell lymphomaObjective:Diffuse large-B-cell lymphoma (DLBCL) is the most common type of lymphoma in adults, accounting for30%to40%of adult non-Hodgkin lymphoma (NHL). DLBCL is an aggressive disease with variable histomorphological, biological and cytogenetic features, as well as clinical outcomes. In the past few years, the introduction of antilymphoma monoclonal antibodies, notably rituximab, in combination with chemotherapy has significantly improved survival outcomes of patients with DLBCL. Nevertheless it is still recrudescent, drug-resistant and even fatal in a part of patients. The malignization of most lymphoma is a complex process with multifactor. In spite of extensive research, the pathogenesis of DLBCL has not been clearly understood. In this study, we performed a gene expression profiling study in DLBCL tissues and tissues from patients of reactive hyperplasia of lymph node by using35k Human Genome Oligo Array and carry on Pathway analysis to probe the relation between some different genes and DLBCL.Material and Methods:1. Specimen collection2. Human Genome Oligo Array (35K)a RNA extraction from tissues of lymph nodeb Reverse transcription and fluorescence labelingc Microarray hybridization and washinge Microarray scanningf Image acquisition and microarray data analysis Results:1. By comparing the gene expression profile of DLBCL and reactive hyperplasia of lymph node tissues, differentially expressed genes were screened (Ratio≥2or≤0.5). The first gene chip included955up-regulated genes and1019down-regulated one. And the second one included587up-regulated genes and1318down-regulated genes.2. The result of KEGG, BioCarta and GenMAPP Pathway analysis showed that the different genes are including signal transduction-related genes, oncogenes and proto-oncogenes, immunodeficiency and immunoregulation-related genes, tumor-related genes, apoptosis-related genes, cytokine receptor-related genes, metabolism genes, cell cycle-related genes, binding protein-associated genes, proteasome-related genes, cytotoxicity-related genes, complement and coagulation cascade-related genes, antigen processing and presentation-related genes, cell adhesion molecule-related genes, et al. Although most genes exert differenent biological functions, they also have connection with each other to form a complicated network and contribute to multiple life activities and the process of diseases.3. MTDH and several Wnt/(3-catenin signaling-associated genes were upregulated, such as LEF1,CCND2,CCND1,et al.Conclusions:1. DLBCL is an aggressive disease with variable histomorphological, biological and cytogenetic features, as well as clinical outcomes. The malignization of most lymphoma is a complex process with multigene and multifactor.2. In this study, we performed a gene expression profiling study in DLBCL tissues and tissues from patients of reactive hyperplasia of lymph node by using35k Human Genome Oligo Array. A serial of differentially expressed genes with variable function are found and these genes may paly a significant role in the pathogenesis of DLBCL.3. MTDH and Wnt/β-catenin pathway are probably involved in the pathogenesis of DLBCL.Part Ⅱ Expressions of MTDH,Wnt/p-catenin pathway in diffuse large B-cell lymphoma and their clinical significanceObjective:MTDH is a new gene that was first cloned several years ago. Studies show that MTDH, involved in aberrant proliferation, survival, and increased migration, invasiveness, and metastasis of tumor cells, has been demonstrated as a potentially crucial mediator of various types of human malignancies. MTDH promotes tumor progression by modulating multiple oncogenic signaling pathways, such as NF-κB, PI3K/Akt and Wnt/β-catenin pathways. Wnt/β-catenin pathway plays an important role in progression of several subtypes of lymphoma. P-catenin is the chief downstream effector of Wnt/p-catenin pathway that migrates to the nucleus and mediates the activation of Wnt/β-catenin pathway. To investigate the expressions of MTDH and Wnt/β-catenin signaling pathway in DLBCL, DLBCL tissues and tissues from patients of reactive hyperplasia of lymph node were collected. We detected the mRNA and protein levels of MTDH and β-catenin in DLBCL tissues and their counterparts, as well as the localization of β-catenin, and examined the correlation between the expressions of MTDH,β-catenin protein and the clinical features of DLBCL in order to illuminate the role of MTDH and Wnt/β-catenin pathway in the pathogenesis of DLBCL and seek potential therapeutic target in the DLBCL patients.Material and Methods:1. Specimen collection2. RNA extraction and real-time quantitative RT-PCR3. Protein extraction and Western Blot4. Immunohistochemistry5. Statistical analysisResults:1. MTDH,β-catenin are overexpressed in DLBCL tissues. DLBCL showed higher expression of MTDH and β-catenin in contrast to reactive hyperplasia of lymph nodes on both the mRNA and protein levels (P<0.0001). The results showed high expression of MTDH in76.67%(23/30) DLBCL cases by using immunohistochemical analysis. Morevoer, nuclear β-catenin was observed in46.67%(14/30) DLBCL cases, whereas no inflammatory lymph node tissue showed nuclear accentuation of β-catenin。2. Upregulation of MTDH and nulcear localization of p-catenin was strongly correlated to the clinical staging of patients with DLBCL (IHC).Statistical analyses were done to examine the correlation between the expressions of MTDH β-catenin protein and the clinical features of DLBCL (n=30). There was no correlation between the expressions of MTDH,β-catenin and patient age (P=0.204, P=1.000), gender (P=1.000, P=0.466) or B symptoms (P=1.000, P=0.713). Nevertheless, the expression level of MTDH protein and the nulcear localization of p-catenin were strongly correlated to the clinical staging of patients with DLBCL (r=0.507, P<0.05; r=0.788, P<0.05).Conclusions:1. We detected that MTDH and β-catenin were both overexpressed in DLBCL tissues by using real-time RT-PCR and Western blot analysis.2. Immunohistochemical staining results showed that nuclear β-catenin was observed in46.67%(14/30) DLBCL cases, which demonstrates that Wnt/β-catenin pathway is partly activated in DLBCL.3. Upregulation of MTDH protein and the nulcear localization of β-catenin were strongly correlated to the clinical staging of patients with DLBCL.4. MTDH and β-catenin may contribute to the pathogenesis of DLBCL together.Part Ⅲ Study about the effect of MTDH expression on biological behavior of diffuse large B-cell lymphoma cells and its mechanismObjective:MTDH, involved in aberrant proliferation, survival, and increased migration, invasiveness, and metastasis of tumor cells, has been demonstrated as a potentially crucial mediator of various types of human malignancies. MTDH expression is significantly higher in the cell lines of multiple types of carcinomas. MTDH promotes tumor progression by modulating multiple oncogenic signaling pathways. In this study, the expressions of MTDH and β-catenin were detected in human DLBCL cell lines (LY1and LY8). Studies are ongoing to further clarify the biological impact of MTDH overexpression on DLBCL tumor cells, in which we investigated that the gained function of MTDH through its upregulation induced by TNF-a and the loss function of MTDH through an MTDH knockdown using stable lentivirus expression vectors in DLBCL cells. Finally, the relationship between MTDH and Wnt/β-catenin pathway in the pathogenesis of DLBCL also needs to be determined.Material and Methods:1. Specimen collection2. Cell culture3. MTDH knockdown using stable lentivirus expression vectors in DLBCL cells4. Detection of the infection efficiency of lentivirus by FACScan flow cytometer5. RNA extraction and real-time quantitative RT-PCR6. Peripheral blood mononuclear cells (PBMCs) isolation7. Protein extraction and Western Blot8. Assessment of cell apoptosis by FACScan flow cytometer9. Assessment of cell proliferation using3H-TdR incorporation method10. Statistical analysisResults:1. The protein levels of MTDH and β-catenin were much higher in the human DLBCL cell lines compared with PBMCs from healthy samples. Making use of subcellular protein fractionation and Western Blot, β-catenin was detected in the nuclear lysates in2DLBCL cell lines.2. The increase of MTDH induced by TNF-a could promote the proliferation of2DLBCL cell lines (LY1:5500±630.2v.15393±1603C.P.M, n=3, P<0.05; LY8:12067±1313v.24943±3938, n=3, P<0.05) and inhibit the cell apoptosis (LY1:23.83±1.762v.16.20±0.9849%, n=3, P<0.05; LY8:14.23±0.3512v. 8.63±0.7496%, n=3, P<0.05).3. After treatment with TNF-a, the protein level of total and nuclear P-catenin were elevated in the meantime (P<0.05) following MTDH upregulation.4. MTDH knockdown using stable lentivirus expression vectors could downregulate the expression of MTDH in DLBCL cells by post-transcriptional mechanism.5. MTDH knockdown cells exhibited significantly higher apoptotic rate in comparison to the cells transfected with negative control siRNA (13.11±6.744v.23.43±6.361%, n=3, P<0.05).6. The total, cytoplasmic and nuclear P-catenin protein was all decreased induced by MTDH silencing in LY8cells (P<0.05).Conclusions:1. MTDH and P-catenin were both overexpressed in the human DLBCL cell lines, with p-catenin nuclear localization.2. The increase of MTDH could promote the proliferation of DLBCL cells and inhibit the cell apoptosis.3. MTDH knockdown could enhance cell apoptosis of DLBCL.4. MTDH upregulation promotes translocation of β-catenin to the nucleus, and further activates Wnt/β-catenin signaling pathway.5. MTDH contributes to the pathogenesis of DLBCL mediated by activation of Wnt/β-catenin pathway.