The Effection Of Ebv-miR-BART6-5p On Proliferation And Apoptosis Of EBV Associated Lymphoma Cell

Chapter 1 Bioinformatic analysis of microarrays identified genes and EBV mi RNA in EBV related lymphomaObjective: The aim of this study was to excavate the EBV mi RNA that associated with lymphoma and their potential target genes through microarray expression profiling data analysis. Methods: mi RNA microarray profileGSE52961 and gene expression profile GSE38885 were downloaded from Gene Expression Omnibus(GEO) database and the dysregulated mir RNA/ m RNA were screened with the GE02 R. Then the correlation between the differentially expressed mi RNAs/m RNA and the EBV related lymphoma was investigated through variety bioinformatic methods, including biological process annotation, KEGG pathway enrichment, EBV mi RNA- m RNA interaction analysis.Finally EBV mi RNA were further identified in EBV positive cell lines Raji, Daudi, Farage and EBV negative cell lines Ramos, Pfeiffer by Real-time quantitative PCR. Results: One mi RNA expression microarray dataset and one m RNA expression microarray dataset were conformed to the standard were obtained from the GEO database. Through analysis of the microarray data, we obtein 18 EBV mi RNAs that up regulated and 38 mi RNA that down regulated in EBV positive lymphoma. KEGG pathway enrichment analysis of 18 EBV mi RNA identify multiple pathways such as Wnt signaling pathway, MAPK signaling pathway that associated with lymphoma occurrence and progress.Biological process annotation implicated that the above-described 38 genes involved in a variety of biological process such as lymphocyte differentiation, regulation of transcription.In addition, EBV mi RNA- m RNA interaction analysis showed that the existence of targeted regulatory relationship between the 18 EBV mi RNAs and the 38 genes. In addiction, ebv-mi RBART11-5p, ebv-mi R-BART14-3p, ebv-mi R-BART1-3p, ebv-mi R-BART6-5p, ebv-mi R-BART17-5p, ebv-mi R-BART11-3p regulated more than half of the genes. In the following Real-time quantitative PCR experiment, the relative expression levels of 11 EBV mi RNAs in the EBV positive and negative lymphoma cell lines were coincided with the chips. Conclusions: We found EBV mi RNAs that up regulated in EBV positive lymphoma and their potential target genes through analyzing microarray data. These results provide a guidance in molecular biological experiments. The relative expression level of ebv-mi R-BART6-5p in EBV positive lymphoma cell lines is high and it is in the center position in regulatory network. We will further explore biological function of ebv-mi R-BART6-5p.Chapter 2 The effection of ebv-mi R-BART6-5p on proliferation and apoptosis in EBV relative lymphoma cellObjective:In this part we will explore the ebv-mi R-BART6-5p impact on the biology function of EBV positive lymphoma cell Farage through the experiments in vitro. Methods: In our study, transfection with EBV mi RNA was performed according to the protocol to establish the down regulated ebvmi R-BART6-5p stable cell lines. The ebv-mi R-BART6-5p expression level further identified by real-time quantitative PCR. Cells growth ability were analyzed with the CCK8 assay. Apoptosis was analyzed with Flow cytometry. Detecting the protein levels of Akt, Bax, NF-κB p65 and Iκκβ through western blot. Results: The level of ebv-mi R-BART6-5p was decreased significantly in cells transfected with GV-mi R-BART6-5p down lentivirus(Farage-BART6-5pdown) compared with the negative control(Farage-NC) and Farage, P<0.05. The level of ebv-mi R-BART6-5p was decreased significantly in BART6-5p inhibitor/Farage compared with the negative control(mi R-sh NC/Farage) and Farage, P<0.05. The level of ebv-mi R-BART6-5p was overexpression in BART6-5p mimics/Pfeiffer compared with the negative control(mi Rsh NC/Pfeiffer) and Pfeiffer, P<0.05. The effect of ebv-mi R-BART6-5p on the viability of Farage cell was detected by the CCK8 assay. The cell proliferation was significantly inhibited at 72 h after recombinant lentivirus GV-mi RBART6-5p down infection, P<0.05. The Flow cytometry analysis showed that the apoptosis rate of Farage-BART6-5p-down cells was significantly higher than the other two groups of cells P<0.05. The protein levels of Akt, NF-κB p65 and Iκκβ were increased significantly, P<0.05. Conclusions: Transfecting GVmi R-BART6-5p down lentivirus into the Farage cells can affect cell biology.Chapter 3 The verification of the target gene of ebv-mi R-BART6-5pObjective: We have found the effects of changing expression level of ebvmi R-BART6-5p in EBV positive lymphoma cell line Farage through biological experiments. In this part we will verify the target gene of ebv-mi R-BART6-5p. Methods: In our study, transfection with EBV mi RNAs was performed according to the protocol to establish the down regulation stable cell lines(GVmi R-BART6-5p down/Farage) and NC/Farage. We predict target gene of ebvmi R-BART6-5p by biological software and verified the protein expression of target gene of the cells after transfected ebv-mi R-BART6-5p by Western Blot. Finally through the dual luciferase report system to validate DICER1 exist on the role of ebv-mi R-BART6-5p targets. Results: We predict target gene of ebvmi R-BART6-5p by biological software and choose DICER1 as the target gene. We found that the DICER1 protein expression level of GV-mi R-BART6-5p down/Farage group was higher than the other two groups by Western Blot. The dual luciferase reporter vectors containing 3’UTR of DICER1 were constructed by using Dual-Luciferase Reporter Assay System, and then the relative activity of firefly luciferase was detected to confirm the binding site of ebv-mi RBART6-5p on DICERl. Conclusions: DICERl is the target gene of ebv-mi RBART6-5p and down regulation of ebv-mi R-BART6-5p could up regulate the DICER1.