| Background: Epstein-Barr virus(EBV),also known as human herpes virus 4(HHV4).EBV infection is widespread in the population,with a global adult infection rate of more than90%.EBV is also the first human tumor virus to be discovered and is strongly associated with the development Burkitt’s lymphoma(BL),Hodgkin’s lymphoma(HL),NK/T cell lymphoma(NKTCL),nasopharyngeal carcinoma(NPC)and EBV-associated gastric cancer(EBVa GC).EBV can be divided into two infection patterns: latent infection and lytic infection.The latent infection is the important material basis of its carcinogenesis.EBVa GC was classified betweentypes I and II latent infection,which mainly expressed EBV-encoded small RNAs(EBERs)and EBV Nuclear Antigen 1(EBNA1),and some cases expressed Latent membrane protein 2A(LMP2A).Nuclear Respiratory Factor 1(NRF1)is a powerful transcription factor,and recent literature has shown that NRF1 has an important role in the development of multiple tumors,such as breast cancer,prostate cancer,colorectal cancer and so on.LMP2 A,a product encoded by EBV,has been shown to regulate NRF1,but the biological function of NRF1 in EBVa GC has not been reported.Objective: In order to clarify the regulatory role and molecular mechanism of EBV latent membrane protein LMP2 A on NRF1 expression,to study the biological role of NRF1 in gastric cancer cell lines,and to explore how NRF1 is involved in maintaining EBV latent infection.Materials and methods:(1)LMP2A recombinant expression plasmids were transfected into EBV-negative gastric cancer cell lines MGC803 and HGC27,G418 was added for screening,and the fluorescence efficiency was observed under fluorescence microscopy,when the transfection efficiency reached 90%,cells were collected and total RNA was extracted,and LMP2 AmRNA transcription levels were detected by quantitative Real-time PCR(q RT-PCR)to verify whether the cell lines MGC803-LMP2 A and HGC27-LMP2 A that stably express exogenous LMP2 A are successfully constructed.(2)EBV-positive gastric cancer cell line SNU719 was treated with small interfering RNAs targeting LMP2 A,NRF1 and p65 coding genes,respectively,Western Blot was used to detect the expression of NRF1 and NF-κB pathway related proteins,migration-related proteins and apoptosis-related proteins in SNU719 cells.(3)The recombinant plasmids NRF1 were transiently transfected into EBV-negative gastric cancer cell lines MGC803 and HGC27,respectively,and the recombinant plasmids NRF1 were transiently transfected into EBV-positive gastric cancer cell lines SNU719 and GT38,respectively,follow-up experiments with cells.(4)The transcription levels of NRF1 mRNA in MGC803-LMP2 A and HGC27-LMP2 A cells and BZLF1,BRLF1 and EBNA1 in SNU719 and GT38 cells overexpressed NRF1 were detected by q RT-PCR.(5)Western Blot was used to detect the expression of NRF1 and NF-κB related proteins in MGC803-LMP2 A and HGC27-LMP2 A cells,changes of migration-related and apoptosis-related proteins in MGC803 and HGC27 cellsoverexpressed NRF1,and expression of EBNA1,BZLF1,BRLF1,VCA and gp350 in SNU719 and GT38 cellsoverexpressed NRF1.(6)The EBV-positive gastric cancer cell line SNU719 was treated with BAY11-7085,an inhibitor of NF-κB pathway,at the concentration of 2μM and 4μM,respectively,the expression of NRF1 and NF-κB pathwayrelated proteins was detected by Western Blot.(7)Transwell analysis was used to analyze the effect of NRF1 on cell migration in gastric cancer cell lines.(8)Flow cytometrywas used to analyze the effect of NRF1 on the anti-apoptosisability of cells in gastric cancer.(9)Droplet digital PCR(dd PCR)was used to detect EBV DNA copy numbers in SNU719 and GT38 cells overexpressed NRF1.(10)The binding sites of the Bam HI-Q promoter(Qp)for the NRF1 and EBNA1 promoters were predicted according to the JASPAR website(http://JASPAR.genereg.net/#),and the corresponding Qp wild-type and mutant reporter plasmids were constructed using p GL3-Basic.In addition,p RL-TK was used as control to construct plasmid.To test the effect of NRF1 on EBNA1 expression,wild-type plasmids(p GL3-Basic-Qp-WT)or mutant plasmids(p GL3-Basic-Qp-MUT)were cotransfected with NRF1 plasmids or NC plasmids into HEK-293 T cells.The lysis was harvested 48 h after transfection and luciferase activity was detected using a dual luciferase reporter system to verify whether NRF1 could bind to the promoter region of EBNA1 to promote transcription.Results:(1)Cells transferred to LMP2 A recombinant expression plasmid were screened,and when 80-90% green fluorescence was observed under fluorescence microscopy,cells were collected and q RT-PCR detection was performed,and the results showed that LMP2 AmRNA transcription levels were significantly upregulated in MGC803-LMP2 A and HGC27-LMP2 A cells compared with the control group,indicating that the cell lines MGC803-LMP2 A and HGC27-LMP2 A stably expressing exogenous LMP2 A had been successfully constructed.(2)Compared with the control group,there was no significant difference in NRF1 mRNA transcription level in MGC803-LMP2 A and HGC27-LMP2 A cells.However,Western Blot assays showed upregulated levels of NRF1 and NF-κB pathwayrelated proteins.(3)After the LMP2 A in the EBV-positive gastric cancer cell line SNU719 was interfered,the expression of NF-κB pathwayrelated protein and NRF1 were downregulated.(4)Western Blot test results showed that after SNU719 cells were treated with NF-κB pathway inhibitor BAY11-7085,the expression of NF-κB pathwayrelated protein and NRF1 protein was more significantly downregulated with the increase of concentration.(5)After overexpression of NRF1 in EBV-negative gastric cancer cell lines,cell migration ability and cell anti-apoptotic ability were enhanced compared with the control group.After interfering with NRF1 in EBV-positive cell lines,both the migration ability and anti-apoptotic ability of cells were reduced.(6)After overexpression of NRF1 in EBV-negative gastric cancer cell lines,Western Blot analysis showed that the expression of N-cadherin,ZEB1,Bcl-2 was upregulated,whereas that of E-cadherin,Bax,caspase3 expression was downregulated.After interference with NRF1 in EBV-positive gastric cancer cell lines,the expression of N-cadherin,ZEB1,Bcl-2 was downregulated,whereas the expression of E-cadherin,Bax,caspase3 was upregulated compared with the control group.(7)Western Blot analysis showed that the expression of BZLF1,BRLF1,gp350 and VCA was downregulated,while the expression of EBNA1 was upregulated in EBV-positive gastric cancer cell lines after overexpression of NRF1,compared with the control group,these results suggested that NRF1 could maintain EBV latent infection.(8)The results of q RT-PCR showed that the transcriptional levels of EBNA1 were increased in SNU719 and GT38 cells overexpressed NRF1,while the transcriptional levels of BZLF1 and BRLF1 did not change significantly compared with the control group.(9)JASPAR predicted that two binding sites might bind to NRF1,and dual luciferase reporter experiments showed that NRF1 could promote transcription of EBNA1 promoter.Conclusion:(1)In EBVa GC,EBV latent protein LMP2 A could promote NRF1 expression through NF-κB pathway.(2)The overexpression of NRF1 can promote the migration of gastric cancer cells and enhance the anti-apoptotic ability of gastric cancer cells.(3)NRF1reduces the expression of EBV lytic proteins BZLF1,BRLF1,gp350 and VCA,and contributes to the maintenance of EBV latent infection status by promoting the transcriptional upregulation of the latent protein EBNA1 promoter. |
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