Study On The Mechanism Of Interaction Between LMP2A And CXCR4 To Promote The Development And Progression Of EBVaGC

Epstein-barr virus(EBV)is a member of lymphocytopirus in the herpesvirus family.Human is the host of EBV infection,and the infection rate of adult in the world is more than 90%.EBV is the first human tumor virus discovered.EBV presents two modes of infection: latent infection and lytic infection.Latent infection status is its important carcinogenic basis.According to the differences in the expression of EBV nuclear antigen(EBNA)and latent membrane protein(LMP),EBV-associated gastric cancer(EBVa GC)is classified as type I latent type,mainly expressing LMP2 A and EBNA1.Chemokine receptor 4(CXCR4)is a 7-pass transmembrane G protein-coupled receptor with low or nonexistent expression in normal tissues.Studies have shown that CXCR4 is the most widely expressed chemokine receptor in more than 23 kinds of human cancers,including breast cancer,ovarian cancer,melanoma,colorectal cancer,etc.,affecting the proliferation,migration,invasion,apoptosis and other biological functions of tumor cells.Several studies have shown that EBV-encoded products can affect the biological function of tumor cells by regulating multiple genes and multiple pathways.In this study,we discussed and analyzed the interaction between EBV encoding products and CXCR4,in order to further clarify the mechanism of EBVa GC occurrence and development.Objective: To clarify the mechanism of interaction between EBV-encoded products LMP2 A and CXCR4,and to explore how CXCR4 is involved in maintaining EBV latent infection and the role of CXCR4 in EBV positive cell lines.Methods:(1)LMP2A was overexpressed in EBV-negative gastric cancer cell line SGC7901,CXCR4 was overexpressed in EBV-positive gastric cancer cell line SNU719.,and 1mg/ml G418 was added for screening.The transfection efficiency was observed under fluorescence microscope,and cells were collected when the transfection efficiency reached 90%.Quantitative Real-time PCR(q RT-PCR)was used to verify the results.(2)Small interfering RNAs targeting LMP2 A,CXCR4 and ERK1/2 were used to act on EBV-positive gastric cancer cell lines GT38 and SNU719,respectively.Western Blot was used to detect the expression of EBNA1,LMP2 A,CXCR4,MAPK/ERK pathway related proteins and NF-κB pathway related proteins in GT38 and SNU719 cells after knockdown.(3)q RT-PCR was used to detect the expression of LMP2 A m RNA and CXCR4 m RNA in SGC7901-LMP2 A cells.(4)Western Blot to detect the expression of CXCR4 and NF-κB pathway related proteins in SGC7901-LMP2 A cells and the expression of LMP2 A,EBNA1,BZLF1,MAPK/ERK pathway related proteins and EMT related proteins in SNU719-CXCR4 cells.(5)NF-κB pathway inhibitor BAY-7085 was used to treat EBV-positive gastric cancer cell lines GT38 and SNU719 with 2μM and 4μM,and the proteins under different treatment conditions were extracted.Western Blot was used to detect the expression changes of CXCR4 and NF-κB pathway-related proteins.(6)MAPK/ERK pathway inhibitor PD0325901 was used to treat EBV-positive gastric cancer cell lines GT38 and SNU719 with 2μM and 4μM,and the proteins were extracted under different treatment conditions.Western Blot was used to detect the expression changes of LMP2 A,EBNA1 and MAPK/ERK pathway related proteins.(7)Transwell analyzed the effect of CXCR4 overexpression on cell migration in EBV positive gastric cancer cell lines.(8)Plate clone formation experiment and CCK-8 analyzed the effect of CXCR4 overexpression on cell proliferation in EBV-positive gastric cancer cell lines.(9)Flow cytometry was used to analyze the effect of CXCR4 overexpression on cell cycle and cell apoptosis in EBV positive gastric cancer cell lines.(10)SNU719-CXCR4 cells were treated with PMA/TPA(PKC activator)and divided into the following four treatment groups: SNU719-NC + DMSO group,SNU719-CXCR4 + DMSO group,SNU719-NC + TPA group and SNU719-CXCR4 + TPA group.Total cell protein was extracted and Western Blot was used to detect the expression of BZLF1,LMP2 A and EBNA1.Results:(1)After the recombinant expression plasmids LMP2 A and CXCR4 were transfected and screened for 3-4 weeks,80-90% of the green fluorescent protein was observed under the fluorescence microscope.Cells were collected for q RT-PCR detection,and the results showed that compared with control cells,The transcription level of LMP2 A m RNA was significantly increased in SGC7901-LMP2 A cells and CXCR4 m RNA was significantly increased in SNU719-CXCR4 cells too.The results indicated that SGC7901-LMP2 A and SNU719-CXCR4 cell lines expressing exogenous LMP2 A and CXCR4 were successfully screened.(2)After knocking down the expression of LMP2 A in EBV-positive gastric cancer cell lines,the expressions of CXCR4 and NF-κB pathway related proteins were down-regulated;after knocking down the expression of CXCR4,the expressions of EBNA1,LMP2 A and MAPK/ERK pathway related proteins were down-regulated;after knocking down the expression of ERK1/2,the expression of EBNA1 and LMP2 A were both down-regulated.(3)Compared with control cells,SGC7901-LMP2 A cells could detect increased expression of CXCR4 m RNA.Western Blot analysis showed the expressions of CXCR4 and NF-κB pathway related proteins in SGC7901-LMP2 A cells were increased,and the expressions of LMP2 A,EBNA1,BZLF1,MAPK/ERK pathway related proteins and EMT related proteins were increased in SNU719-CXCR4 cells.(4)After GT38 and SNU719 cells were treated with NF-κB pathway inhibitor BAY-7085,Western Blot results showed that the expression of NF-κB pathway related protein and CXCR4 in the inhibitor group was decreased,and that in the 4μM inhibitor group was significantly lower than that in the 2μM inhibitor group.NF-κB pathway was found to induce CXCR4 expression in a dose-dependent manner.(5)After treatment of GT38 and SNU719 cells with MAPK/ERK pathway inhibitor PD0325901,Western Blot results showed that the expression of EBNA1,LMP2 A and MAPK/ERK pathway related proteins decreased in the inhibitor treatment group,and in the 4μM inhibitor treatment group was significantly lower than the 2μM inhibitor treatment group.It is found that the MAPK/ERK pathway can induce the expression of EBNA1 and LMP2 A in a dose-dependent manner.(6)Compared with the control group,the overexpression of CXCR4 in EBV-positive gastric cancer cell lines promoted the migration and proliferation ability of cells.However,there was no significant regulation of cell cycle and cell apoptosis.(7)Western Blot results showed that the expression of BZLF1 in SNU719-NC +TPA group was significantly higher than the other three groups.In the SNU719-CXCR4 + TPA group,the expression of BZLF1 was lower than that in the SNU719-NC +TPA group.However,the expression level of BZLF1 in the SNU719-CXCR4 + DMSO group was lower than the other three groups.These results indicate that CXCR4 can reverse TPA-induced BZLF1 elevation to a certain extent.Moreover,the expression levels of EBNA1 and LMP2 A were higher in the CXCR4 overexpression group.The above results suggest that CXCR4 can inhibit the expression of BZLF1 and promote the expression of EBNA1 and LMP2 A.Conclusion:(1)There is a positive feedback between the EBV-encoded product and CXCR4,and LMP2 A can activate CXCR4 through the NF-κB pathway.In addition,CXCR4 can be fed back to LMP2 A and EBNA1 through the ERK signaling pathway.(2)CXCR4 can promote the proliferation and migration of EBV-positive cells,but have no significant effect on cell cycle and cell apoptosis.(3)CXCR4 can reduce the expression of immediate early protein BZLF1,and play an important role in maintaining the incubation period of EBV infection.