Ebv-miR-BART19-3p Targets APC To Enhance The Proliferation Of Lymphoma Cells

ObjectiveLymphoma is a malignant tumor that originates from the hematopoietic system,and is classified into two types,non-Hodgkin’s lymphoma(NHL)and Hodgkin’s lymphoma(HL)based on tissue characteristics.Through miRNA microarray scanning,our group found that the expression of ebv-miR-BART19-3p was increased in a mouse model of EBV-induced lymphoma.In previous studies,the biological function of ebv-miRBART19-3p at the cell level has not been explored.EBV-induced lymphoma is a process involving multiple signal pathways,multiple proteins,and mutual regulation with host genes.This study focuses on the influence of which pathway and which protein ebv-miR-BART19-3p passes through on the biological functions of lymphoma cells.Methods1.Detect the expression of EBNA1 and LMP1 in three lymphoma cell lines(raji,daudi,and ramos are all derived from Burkitt lymphoma).2.Detect the expression of ebv-miR-BART19-3p in three cell lines by RT-PCR;select two cell lines with high and low expression of ebv-miR-BART19-3p for follow-up experiments.3.Through transfection to overexpress and inhibit the expression of ebv-miR-BART19-3p in cells,observe which phenotypic changes occur in the cells;construct a stable and high-expressing ebv-miR-BART19-3p cell line in Daudi cells through lentivirus.Transfection of miRNA antagomir with ribo FECT CP transfection reagent downregulates the expression of ebv-miR-BART19-3p,and observes the phenotypic changes of cells after transfection.4.Analyze the possible target genes of ebv-miR-BART19-3p by consulting the literature and the biometric analysis website(https://bibiserv.cebitec.unibielefeld.de/rnahybrid).5.Directly verify the protein expression of the targeted gene by Western blot.Results1.Extract RNA from three cell lines(Daudi,Ramos and Raji were derived from Burkitt’s lymphoma),perform PCR,and perform gel electrophoresis through PCR products to confirm that the three cell lines are infected with EBV.The results show that Raji cells are strongly positive,while Ramos and Daudi cells are almost not.Express EBNA1 and LMP1;we will select one strain from Ramos and Daudi cells as the overexpression experimental group.2.The total miRNA of three cell lines was extracted,and the expression of ebv-miRBART19-3p in the three cell lines was confirmed by relative fluorescence quantitative PCR.The results showed that ebv-miR-BART19-3p was highly expressed in Raji cells with statistical differences(P<0.001),it was not amplified in Daudi and Ramos cells.3.Try to construct ebv-miR-BART19-3p stable and high-expressing cell lines in Daudi and Ramos cells;Ramos cells are sensitive to viruses and grow poorly after transfection.Finally,Daudi is selected to construct stable transgenic strains;by relative fluorescence quantitative PCR It was confirmed that ebv-miR-BART19-3p was relatively highly expressed in Daudi cells after transfection(P<0.001);CCK-8 cell proliferation test confirmed that ebv-miR-BART19-3p promoted the proliferation of Daudi cells.4.Transiently transfected miRNA antagomir in Raji cells interfered with the expression of ebv-miR-BART19-3p;by flow cytometry,cell apoptosis increased after transfection,cell cycle slowed down,and CCK8 cell proliferation experiments proved transfection The proliferation of Raji cells decreased afterwards.5.Through literature search and RNAHYBRID database analysis,ebv-miR-BART19-3p binds to the seed binding site of APC;through KEGG database and cell signaling technology website,we found that the protein complex involved in APC inhibits the expression of β-catenin,we Initially,the stably transfected Daudi cells were used to detect the expression of APC,and the results were in line with expectations.6.Through Western blot detection,it was found that after interference with ebv-miRBART19-3p,the expression of APC in Raji cells increased,while the expression of the downstream target gene β-catenin of APC decreased,which reduced the nuclear accumulation of β-catenin and inhibited Proliferation of cells and promote apoptosis.Conclusions1.Overexpression of ebv-miR-BART19-3p in Daudi cells promotes cell proliferation and inhibits apoptosis.2.Interfering with the expression of ebv-miR-BART19-3p in Raji cells inhibits cell proliferation and promotes apoptosis.3.ebv-miR-BART19-3p promotes the accumulation of β-catenin and induces cell proliferation through targeted inhibition of APC.