| Objective To study the effects of latent membrane protein 1(LMP1)on the proliferation and cell cycle of human B-lymphoma cell line,and its regulation of proteins related to immune function,and explore the possible mechanism.Methodology(1)Connect the LMP1 sequence into the pcDNA3.1-EF1a-mcs-3flag-CMV-EGFP vector,and transfect the Ramos cells by electroporation,Ramos cells stably expressing LMP1 were screened by geneticin(Geneticin,genetic G418).RT-PCR and Western Blot were used to confirm the expression of LMP1 in Ramos cells after transfection.(2)Use the CCK8 method to draw the cell growth curve and detect the cell proliferation ability.Flow cytometry was used to detect the cell cycle distribution,and qRT-PCR was used to detect the expression of Bax and Bcl-2 mRNA.(3)Western Blot and immunofluorescence method were used to detect the quantification and localization of interferon regulatory factor 7(IRF7)in cells,and co-immunoprecipitation preliminarily screened the interacting proteins of LMP1 in Ramos cells.Results(1)The pcDNA3.1-EF1a-mcs-3flag-CMV-EGFP-LMP1 vector was constructed and Ramos cells expressing LMP1 stably were selected.(2)After Ramos cells over-expressed LMP1 stably,there was no significant change in the time for the cells to enter the incubation and exponential growth phase,but the proliferation ability was enhanced(P <0.05).Flow cytometric analysis showed that compared with the control group,the ratio of G0 / G1 phase cells in the LPM1 overexpression group was reduced to(20.51 ± 1.834)%,while the control group was(26.03 ± 0.45)%,P <0.05;The proportion of cells in G2/M phase increased by(15.12 ± 0.85)%,and that of the control group was(8.533 ± 1.053)%,P <0.05.There was no significant change in the expression of Bax mRNA(1.027 ± 0.163,P> 0.05),but the expression of Bcl-2 mRNA was up-regulated(1.46 ± 0.111,P <0.05),Bcl-2 / Bax was up-regulated(1.436 ± 0.143,P <0.05).(3)After LMP1 overexpression,the total expression of IRF7 in the cells was up-regulated(1.179 ± 0.044,P <0.05).Immunofluorescence showed that IRF7 was distributed in the nuclei and cytoplasm of both groups,and there was no significant difference in the distribution of IRF7.Co-immunoprecipitation combined Mass Spectrometry and search analysis showed that LMP1 and Glutathione S-transferase P(GSTP1),Heterogeneous nuclear ribonucleoproteins A1(HNRNPA1),and nuclear Heterogeneous nuclear ribonucleoproteins A2 / B1(HNRNPA2B1),heterogeneous nuclear ribonucleoproteins C1 / C2(HNRNPC1C2),serine / arginine-enriched shear factor(Serine / arginine-rich splicing factor 1,SRSF1)has interaction relationship(Sequence coverage> 30%,socre> 20).Conclusion(1)After Ramos cells overexpress LMP1 stably,there is no significant change in the time for the cells to enter the incubation period and exponential growth phase,but the proliferation ability is enhanced,the proportion of G0 / G1 phase cells is reduced and the proportion of G2 / M phase cells is increased;there was no significant change in Bax mRNA expression.,the expression of Bcl-2 mRNA and Bcl-2 / Bax ratio was up-regulated,suggesting that LMP1 can promote the proliferation of Ramos cells,affect the cell cycle distribution and regulate apoptosis-related genes.(2)After LMP1 is overexpressed,the total expression of IRF7 in the cells is up-regulated.Immunofluorescence shows no significant change in the distribution of IRF7,suggesting that LMP1 can upregulate the overall expression of IRF7 to a certain extent,and IRF7 may participate in cell proliferation induced by LMP1 overexpression.(3)Co-immunoprecipitation combined with mass Spectrometry and search analysis showed that LMP1 interacts with GSTP1,HNRNPA1,HNRNPA2B1,HNRNPC1 / C2,and SRSF1,but the nature of the interaction relationship needs to be determined. |
PDF Full Text Request