Analysis And Study Of Gene Expression Profile And MiRNA Expression Profile In EBV-Induced Lymphomas

Background and objective: The relationship between EB virus infection and lymphomagenesis is very close. The lymphoid cell lines were used to study the transformation effect of Epstein Barr virus(EBV) on lymphocyte, but the situation of EBV-induced lymphoma in vivo cannot be comprehensively and objectively reflected with this model in vitro. This study intends to further identify the molecular characterization and clonality of the EBV-induced tumor in the model. Then by using human whole genome microarray and mi RNAs expression profile chip, the differentially expressed genes and mi RNAs were detected between EB virus-induced lymphoma of SCID mice in vivo and the corresponding "normal" lymphocytes. Combined with bioinformatics analysis, the key genes and critical mi RNAs were screened in the process of EB virus-induced lymphomagenesis and describe its regulatory network, and the molecular mechanisms of the Epstein Barr virus associated lymphomagenesis was tried to explained.Methods: lymphocytes were isolated from six healthy adult fresh blood samples, and inoculated into twenty two SCID mice and injected with EB virus, and EBV induced lymphoma model was established. The molecular pathologic features of the tumors were identified by immunohistochemistry, and the monoclonal proliferation of the tumors was detected by Ig H rearrangement and capillary electrophoresis. Agilent human whole genome expression microarray and Exiqon mi RNAs chip were used to detect the differentially expressed genes and differentially expressed mi RNAs between normal human lymphocytes and EBV associated lymphoma. And real-time PCR was used to verify the results of the chip. The differentially expressed genes and mi RNAs were screened by using three different molecular screening(LIMMA, BRB-Random variance model and SAM) software and reassume the intersection at the same time. The PCR real-time method was used to verify the results of the chips. The expression of candidate key gene PBK in EBV+ lymphoma cell line was detected by si RNA interference, and the viability of cells and the ability of clone formation in vitro were detected. Basis on this, the results of gene-chip and mi RNAs-chip were integrated and the diff mi RNAs-diff genes regulation network was drawn. By calculating the enrichment degree, the key different genes and their different mi RNAs in the integrated network would be obtained. The dual luciferase reporter gene system was used to verify the relationship between the ebv-mi R-BART16 and its predicted target gene.Results: 1. Establishment of EBV-induced lymphoma model and its molecular pathologic characteristics The normal lymphocytes were inoculated in SCID mice, through peritoneal primary vaccination site injection of EBV for tumor induction, 6 cases induced tumor were obtained. The induced tumors were found in the mediastinum or abdominal cavity of SCID mice. Microscopic observation exhibited tumor cells that were large and had a plasmablastic, centroblastic or immunoblastic-like appearance, and its pathological typology belongs to diffuse large B cell lymphoma. Immunophenotyping assays showed the induced tumors were LCA-positive, CD20/CD79a-positive(markers of B cells) and CD3/CD45RO-negative(markers of T cells). A human-specific Alu sequence could be amplified by Alu-PCR. This confirmed that induced tumors were B-cell lymphomas originating from the transplanted human lymphocytes rather than mouse cells. EBER in situ hybridization detected positive signals in the nuclei of the tumor cells. Expression of EBV-encoded LMP1, EBNA-1 and EBNA-2 in the tumors was significantly positive. PCR-based capillary electrophoresis analysis of Ig H gene rearrangement revealed a monoclonal peak and single amplification product in all 6 cases of induced tumors. 2. Bioinformatics analysis of differentially expressed genes Gene chip was used to detect the different genes between lymphoma tissue induced by EBV in SCID mice and the corresponding “normal” lymphocyte. By bioinformatics analysis, 202 different expression genes were obtained, of which 44 genes were up-regulated and 158 genes were down-regulated in the tumor tissue. GO analysis and pathway analysis on the gene chip results revealed enrichment in the highest degree of GOs, including signal transduction, cell growth; Enrichment of the highest pathway was cell cycle, cell proliferation and tumor immune escape. Differentially expressed 202 genes were matched to the HIPPIE database, and PPI network diagram was constructed. RS(Rank Score) values of these genes in the network were calculated. The top 10 differential expression genes in the molecular networks were selected based on the analysis of the transcriptional group: TOP2 A, UHRF1, HIST2H2 BE, PHGDH, VCL, IGF1 R, FOS, SNAI1, PBK and RNF144 B. These genes are involved in cell proliferation, differentiation, immune regulation and immune escape and other signaling pathways. Among these genes, the expression of TOP2 A, UHRF1, PHGDH and PBK genes are upregulated in the induced lymphoma, while the expression of HIST2H2 BE, VCL, IGF1 R, FOS, SNAI1 and RNF144 B genes are down regulated in the inducted lymphoma. After the expression of PBK gene in lymphoma cells Daudi was interfered with si RNA technique, the viability of cells was decreased, and the ability of clone formation in soft-agar was reduced. 3. Differentially expressed mi RNAs biological information analysis mi RNA microarray was used to detect the expression of mi RNAs between lymphoma tissue induced by EBV in SCID mice and the corresponding “normal” lymphocyte. The results identified 29 distinct human mi RNAs, of which 25 were down-regulated and 4 were up-regulated; Meanwhile 3 EB virus mi RNAs were identified(ebv-mi R-BART16, ebv-mi R-BART8* and ebv-mi R-BART19-3p). 4. Joint analysis of differentially expressed genes and differentially expressed mi RNAs Based on the relationship between the differential expression of mi RNAs(29 human mi RNAs + 3 EBV mi RNAs) and their target genes, the diffmi RNA-diffgene integrated regulatory network of EBV-induced lymphoma were constructed combined with the differentially expressed genes in the interaction of PPI network. Through the calculation of enrichment degree, we found that mir-23 a, mi R-26 a, mir-26 b, respectively, hsa-mi R-130 a and hsa-mi R-222 were key mi RNAs in the integration of control network and key target genes were AURKA, PBK, IL1 B and EGF, in which PBK and AURKA were up-regulated, and IL1 B and EGF were down-regulated in lymphoma tissue. Targeting analysis between differential expression of EB virus mi RNAs and host cell gene showed that there were genes in the difference of gene chip that can be directly regulated by ebv-mi RNA such as RYBP and caspase-6. These two genes are closely related to the regulation of apoptosis, in particular, caspase-6 is a member of the caspase family, and is one of the executive molecules of apoptosis. Luciferase report gene system was used to verify the target effect of ebv-mi R-BART16 on RYBP and caspase-6. The results show that ebv-mi R-BART16 can inhibit expression of both RYBP and caspase-6.Conclusions: 1. This study successful established model of human B cell lymphoma in vivo induced by Epstein Barr virus in SCID chimeras’ mouse. Lymphoma tissue originated from human B lymphocytes and tumor cells showed characteristics of monoclonal proliferation. Histopathological type of induced lymphoma is diffuse large B cell lymphoma. 2. The differential gene expression profile between lymphoma induced by EBV and corresponding “normal” lymphocytes was constructed. Total 202 differentially expressed genes were screened by bioinformatics analysis, including 44 up-regulated genes and 158 down-regulated genes. These genes mainly involved in cell proliferation and differentiation, immune regulation and immune evasion. It suggests that EBV-induced lymphomagenesis is a complex process involving multiple genes, pathways, and mutual interaction of viral and host gene. Genes including TOP2 A, UHRF1, HIST2H2 BE, PHGDH, VCL, IGF1 R, FOS, SNAI1, PBK and RNF144 B etc. may be the candidate key molecules in the EBV induced lymphomas. 3. Differential mi RNAs expression profile of EBV-induced lymphomas was obtained. A total of 29 distinct human mi RNAs differences and 3 up-regulated EB virus mi RNAs were identified. 4. The differential gene regulation module of EBV-induced lymphoma were obtained through bioinformatics analysis, and integration network of m RNAs-mi RNAs interaction was constructed; Through complex molecular regulatory networks, EBV could regulate cell cycle, mediate immune escape, promote cell proliferation and inhibit apoptosis signaling to induce lymphomagenesis. 5. Ebv-mi R-BART16 can inhibit the expression of RYBP and caspase-6, and may have the ability to inhibit apoptosis.