Study On The Invasion Of Diffuse Large B Cell Lymphoma Mediated By Metadherin

Diffuse large-B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma (NHL), and accounts for 30% to 40% of adult NHL. DLBCL can arise out of lymph node or extranodal tissus, or be transformed from low-grade lymphoma, including chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), marginal zone lymphoma(MZL) and follicular lymphoma(FL) and so on. As an aggressive behaving lymphoma, DLBCL is a heterogeneous group of tumors, with different cytogenetic abnormalities, histomorphological features, clinical characteristics and prognosis. The survival outcomes of patients with DLBCL are significantly improved by initial chemotherapy composed of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Nevertheless nearly 40% of DLBCL patients will eventually die of refractory, relapsed disease. Therefore, studies of biological diversity and invasion mechanism of DLBCL play important roles in the discovery of novel therapeutic targets, optimization of front-line therapy and improvement of prognosis.Metadherin(MTDH) has emerged in recent years as a novel oncogene, which is also known as Astrocyte elevated gene-1 (AEG-1). It was first identified and cloned in primary human fetal astrocytes after human immunodeficiency virus-1 (HIV-1) infection. Afterwards, it was cloned in metastatic breast cancer, and based on its promoting the adhesion of mammary tumor cells to lung, it was named MTDH. The full-length MTDH/AEG-1 cDNA includes 3611bp, and is located at 8q22, which encodes a single-pass transmembrane protein, including 582 amino acid, with molecular weight of 64 kDa. So far, plenty of studies have demonstrated that the expression of MTDH is increased in diverse cancers, such as breast cancer, melanoma, hepatocellular carcinoma(HCC), lung cancer, prostate cancer, renal cell carcinoma, colorectal cancer, glioma, gallbladder carcinoma, ovarian cancer, lymphoma and so on. Further studies confirm that MTDH can regulate cancer proliferation, angiogenesis, invasion, metastasis, and chemoresistance by a group of signaling pathways, including PI3K/Akt, nuclear factor-KB (NF-κB) and Wnt/β-catenin.Recently, it has been found that epithelial-mesenchymal transition (EMT) is critically associated with the local invasion and distant metastasis of tumor. In the process of EMT, cancer cells lose the adhesive ability along with acquiring the properties of invasion and migration. It was shown that tumor cells lost epithelial phenotype markers for example E-cadherin and acquired mesenchymal markers such as cytoskeletal proteins:vimentin and β-catenin, with the upregulation of nuclear expression of several transcription factors (Snail, Twist and ZEBl,et al). EMT plays a vital role in promoting invasion and migration of tumor cells, and pertains to tumor development, it is confirmed in breast cancer,, lung cancer, HCC, head and neck squamous cell carcinoma (HNSCC), osteosarcoma and so on, that MTDH is able to promote the metastasis of tumor by inducing EMT of tumor cells. The expression of MTDH and EMT biomarkers are closely associated with differentiation degree, clinical stage and prognosis of cancers.MTDH promote the EMT of cancers by regulating multiple signaling pathways, of which Wnt/β-catenin acts a significant part. Wnt/β-catenin pathway play important roles both in embryonic development and maintenance of tissues in adults. The activation of Wnt/β-catenin pathway involves in the pathogenesis of divers carcinomas. The pivot between EMT and Wnt pathway appears to reside in P-catenin which potentially couples loss of cell adhesion to the activation of Wnt pathway if diverted from the cytoplasm to the nucleus. DLBCL belonging to highly aggressive lymphoma is particularly liable to extranodal involvement. Whether there is an EMT-like process in DLBCL and the relationship between MTDH and EMT in the invasion of DLBCL have not been reported yet. For the first time, our study demonstrates the expression of MTDH and EMT in DLBCL and the relationship between them in the invasion of DLBCL, by using immunohistochernistry, cellular biology and gene silencing technologies. These findings may contribute to investigation on promising novel biomarkers and therapeutic targets in DLBCL.PartⅠ Expressions of MTDH^ EMT biomarkers in diffuse large B-cell lymphoma and clinical significance of MTDH expressionObjective:DLBCL is an aggressive lymphoma with tremendous heterogeneity. Nevertheless the invasion of DLBCL has not been clearly understood. MTDH has emerged in recent years as a novel oncogene. Plenty of studies have demonstrated that the expression of MTDH is increased in diverse cancers and it can regulate cancer proliferation, angiogenesis, invasion, metastasis, and chemoresistance by a group of signaling pathways, including PI3K/Akt, nuclear factor-KB (NF-κB) and Wnt/p-catenin. EMT is critically associated with the local invasion and distant metastasis of tumor. In the process of EMT, biologic behaviors and biomarkers of cancer cells will change. MTDH is able to promote the metastasis of cancer by inducing EMT of cancer cells. For study the expressions of MTDH and EMT in DLBCL, we detected the protein levels of MTDH and EMT biomarkers (vimentin and ZEB1) in DLBCL cell lines (LY1 and LY8) by Western blot. After that, we collected DLBCL and reactive hyperplasia of lymph node tissues, detected the expression of MTDH in tissues of both groups, and analyzed the correlation between MTDH expression and the clinical parameters of DLBCL patients for exploring the role of MTDH in the invasion of DLBCL.Material and Methods:1. Specimen collection and peripheral blood mononuclear cells (PBMCs) isolation2. Cell culture3. Protein extraction and Western blot4. Specimen and clinical parameters collection4. Immunohistochemistry5. Statistical analysisResults:1. The protein levels of MTDH, vimentin and ZEB1 in DLBCL cell lines (LY1 and LY8) were higher compared with the PBMCs from healthy samples (P<0.05). The target band of MTDH located in 64 kDa by Western blot.2. Significantly elevated levels of MTDH were observed in DLBCL tissues, whereas the protein expression of MTDH was barely detectable in the reactive hyperplasia of lymph node tissues. The results showed that in DLBCL patients only one case scored zero, low expression of MTDH accounted for 40.0%(12/30) and high expression of MTDH accounted for 60.0%(18/30). Moreover, the stains of MTDH were mainly found in cytoplasm, accidentally in the nucleus.3. Statistical analyses were done to analyzed the correlation between MTDH expression and the clinical parameters of DLBCL patients by immunohistochemistry. There was no relevance between the expression of MTDH and patient age (P=0.458), gender (P=0.284) or LDH (P=0.249). However, the expression of MTDH was positively associated with clinical staging (P=0.04), B symptoms (P=0.004) and risk stratification (P=0.033).Conclusions:1. We detected that MTDH, vimentin and ZEB1 were overexpressed in DLBCL cell lines by utilizing Western blot,2. Immunohistochemistry results showed that high expression of MTDH accounted for 60.0%(18/30), and the stains of MTDH were mainly found in cytoplasm, accidentally in the nucleus.3. The expression of MTDH was positively associated with clinical staging, B symptoms and risk stratification of patients with DLBCL.Part Ⅱ The effect of MTDH expression on invasion of diffuse large B-cell lymphoma and its mechanismObjective:It is confirmed in a variety of cancers that MTDH promote the metastasis of cancer by regulating EMT of cancer cells. The expressions of MTDH and EMT biomarkers are closely associated with differentiation of carcinoma, clinical staging and prognosis. MTDH induces EMT of cancer cells by diverse signaling pathways, such as PI3K/Akt, NF-kB, Wnt/(3-catenin and MAPK, to regulate the invasion of cancers. In this study, we silenced the expression of MTDH by stable lentivirus vectors, then detected the changes of protein levels of vimentin and ZEB1, as well as the changes of invasion of DLBCL cells. In this way, we further investigated if there was an EMT-like process in DLBCL and the mechanism of MTDH in regulating the invasion of DLBCL cells.Material and Methods:1. Cell culture2. Silence the expression of MTDH in DLBCL cells by stable lentivirus vectors3. Detection of the transfection efficiency of lentivirus by flow cytometer4. Protein extraction and Western Blot5. Trans well invasion and migration assay6. Statistical analysisResults:1. The silence of MTDH by stable lentivirus vectors downregulated the expression of MTDH in DLBCL cells.2. The protein levels of vimentin and ZEB1 were downregulated in MTDH knockdown DLBCL cells (P<0.05).3. Transwell test results exhibited the migration and invasion decreased in MTDH knockdown DLBCL cells (P<0.05).Conclusions:1. There was an EMT-like process in DLBCL.2. MTDH knockdown could downregulate the expression of vimentin and ZEB1 in DLBCL cells.3. Inhibition of MTDH expression could decrease the migration and invasion of DLBCL cells.4. MTDH regulated the invasion of DLBCL may be mediated by activation of Wnt/β-catenin pathway.