The Effect And Mechanism Of EBI3 On Diffuse Large B-cell Lymphoma

Objectives: In view of a large number of studies found that EBI3 is expressed in lymphomas,and most patients have poor prognosis,the basic research on EBI3 immunoregulatory mechanisms for tumors is less,and the immunosuppressive mechanism of EBI3 on tumors has not been elucidated.To establish an animal model of diffuse large B-cell lymphoma and conduct in vivo experiments to further explore the role of EBI3 in diffuse large B-cell lymphoma.This experiment was a stable cell line with high expression and low expression of EBI3 in mouse B-cell lymphoma cell line A20.Methods:Part 1(1)A20 cells were cultured in vitro and the total protein and total RNA were extracted when the cells were in logarithmic growth phase.RT-qPCR and Wester blot experiments were performed to verify the expression level of EBI3 in A20 cells;(2)Design of sg RNA olige primers for EBI3.According to the http://www.ncbi.nlm.nih.gov/ website to determine the EBI3 CDs region gene sequence,the sg RNA sequence design reference http://crispr.mit.edu/ website.According to the website analysis feedback,choose the sequence with higher score.(3)Construction of lenti CRISPR v2-EBI3.Linearization of the lenti CRISPR v2 vector was first performed,followed by phosphorylation of oligo with T4 PNK and gradient annealing to form the dimer.The sg RNA dimer was then ligated to the v2 linear vector and subjected to competent transformation plating to pick a monoclonal colony.Shake the bacteria,extract the plasmid,and send the sequencing verification.(4)Lentivirus transfection system infected A20 cells.1.First culturing293 T cells in vitro,the virus can be packaged when the cells are in logarithmic growth phase;2.The packaged virus can be used to infect A20 cells,and the virus supernatant is added every other day for 3 days;3.The purine is added at a suitable concentration.Screening positive cells.(5)RT-PCR and Western blot experiments were used to verify the changes of EBI3 expression in RNA and protein levels in A20 cells before and after transfection.(6)Construction of EBI3 high expression lentiviral plasmids.1 Firstly,the CBI of EBI3 gene was found on NCBI,then mouse-derived EBI3 gene 2was ordered on ORIGEN 2 PCR was used to amplify the target gene EBI3;3DNA purification kit was used to recover and purify the target gene fragment;4 will be recovered The target gene fragment was digested,ligated,transformed,and extracted with the lentiviral vector PCDH-CMVMCS-EF1-puro.The plasmid was sent for sequencing and verified.(7)The recombinant EBI3 high expression plasmid was transfected into A20 cells by a lentivirus transfection system.(8)The transfected cells were verified by RT-qPCR and Wester blot experiments for high expression of EBI3.(9)All data are mean ± standard deviation.The statistical software used was SPSS17.0.The analysis method was analysis of variance(ANOVA)and independent sample t test.P<0.05 has statistical significance.Part 2(1)immunohistochemical detection of the expression of EBI3 in the pathological tissue samples of 43 patients with diffuse large B-cell lymphoma;(2)The data is count data.The statistical software used is SPSS17.0.The analysis method is Chi-square test or Fisher’s exact test.P<0.05 has statistical significance.Results:Part 1(1)RT-qPCR and Western blot detection of K562,A20,U266,R8226,MMIS several blood system tumor cells found that the expression of EBI3 in A20 cells was relatively high,P< 0.05 difference was statistically significant.EBI3 low expression cell lines can be established by CRISPR technology,and high expressing cell lines can be established by lentiviral recombinant plasmids.(2)The sequencing results of the recombinant plasmid lenti CRISPR v2-EBI3 showed that the position and orientation of the inserted sequence were correct,and the recombinant plasmid was successfully constructed.(3)The effect of EBI3 knockout detected by Western blot and RT-qPCR showed that compared with A20 wild type cells,EBI3 knockout of A20 cells(hereinafter referred to as A20-EBI3-KO)did not detect EBI3 protein expression.P<0.05.Compared to wild-type A20 cells,EBI3 mRNA expression was decreased in A20-EBI3-KO cells,P< 0.05.It was shown that the A20 cell EBI3 knockout stable strain was successfully constructed.(4)Western blot and RT-qPCR detection of EBI3 gene overexpression showed that compared with A20 wild type cells,high expression of EBI3 gene A20 cells(hereinafter referred to as A20-EBI3-High)detected high EBI3 protein expression(P<0.05)Compared with wild-type A20 cells,the expression of EBI3 mRNA in A20-EBI3-High cells was increased(P<0.05),indicating that the high-expression and stable strains of EBI3 gene were successfully constructed in A20 cells.Part 2Immunohistochemical analysis of 43 specimens of DLBCL revealed that1 case(2.3%)of patients expressed EBI3.Conclusion:(1)The low-expression EBI3 cell line and EBI3high-expression cell line in mouse B-lymphocytic lymphoma cell A20 were successfully constructed,and follow-up experiments can be continued.(2)The exact relationship between EBI3 and diffuse large B-cell lymphoma could not be found.