| Background: Epstein-Barr virus(EBV),belongs to human herpesvirus 4,infects more than 90% of adults worldwide,and its latent infection has been closely associated with many human tumors,known as the DNA tumor virus.EBV genome can be detected in almost all EBV-related tumors.The latent infection of EBV in host cells is an important carcinogenic basis.EBV participates in the development of EBV-related tumors by influencing the gene expression and biological behavior of infected cells through its latent gene products.EBV associated gastric carcinoma(EBVaGC)is considered to be a subtype of gastric carcinomas and has the largest number of EBV-related malignancies incidence.Compared with the EBV-negative gastric carcinoma(EBVnGC),it shows characteristic clinicopathological features and unique oncogenic mechanisms.However,the pathogenesis is not fully understood.EBV can participate in many signal pathways via its infection products,such as NF-κB,JNK,STAT3,PI3K/AKT.EBV induces cell transformation and proliferation,inhibits cell differentiation and apoptosis through regulating the expression of host cells and viral genes.Transcription factor NF-κB,is an important regulator closely related to lymphocyte proliferation,immune response,inflammation and cancerigensis.Constitutive activation of NF-κB signal pathway can induce cell proliferation and inhibit tumor cell apoptosis by up-regulating the expression of its downstream molecules.The important role of NF-κB signaling pathway in tumorigensis and development has been elucidated in many EBV-associated diseases,but its specific mechanism in EBVaGC is still unclear.Objective: 1 The activation of the classical NF-κB signaling pathway in EBVaGC and EBVnGC by detecting the expression and subcellular localization of NF-κB signaling pathway-related molecules in gastric cancer cell lines and tissues.2 To explore the specific mechanism of EBV activating the classical signal pathway of NF-κB through latent infection.3 The effects of NF-kappa B signaling pathway on the proliferation and apoptosis of EBV-positive gastric cancer cells and the expression of LMP1 and LMP2 A were detected and analyzed.Materials and Methods: 1 Total RNA and total protein were extracted from EBV-negative tumor cell lines(SGC7901,BGC823,HGC27),EBV-positive tumor cell lines(GT38,GT39,SNU719).The expression of NF-κB signaling pathway-related molecules(NFKBIA,TRAF1/2/3/6)was detected by real-time fluorescence quantitative PCR(qRT-PCR)and Western Blot;The subcellular localization of p65 protein in gastric cancer cell lines was measured by immunofluorescence technique,and the binding ability of NF-κB transcription factor to DNA in gastric cancer cell lines was detected by ELISA,and the activation of NF-κB signaling pathway in gastric cancer cell lines was judged comprehensively.2 Immunohistochemical technique was used to detect the expression and subcellular localization of IκBα and p65 in EBVaGC and EBVnGC.3 EBVaGC cell line SNU719 and EBVnGC cell line SGC7901 were treated with different concentrations of BAY11-7082,an inhibitor of NF-κB signaling pathway.CCK-8 was used to detect the cell survival after 24 hours.4 Flow cytometry and Annexin-FITC kit were used to detect the apoptotic effect of 2.5μM,5μM and 10μM of NF-κB signaling pathway inhibitor BAY11-7082 on EBVaGC GT38 12 hours later.Western blot was used to detect the expression of apoptosis-related proteins(Bax,Bcl-2,Cleaved Caspase-3)and IκBα,p-IκBα at different concentrations and at different treatment times.5 The expression of ZEB1 was detected by adding BAY11-7082,an inhibitor of NFκB signaling pathway,to GT38 EBV-positive cells.In this experiment,total protein was extracted from GT38 cells at concentrations of 2.5μM,5μM and 10μM,respectively.The expression of ZEB1 protein was detected by Western blot.6 The promoter region of NFKBIA gene was amplified with BGS specific primers.There were 82 CpG loci in this region.The amplified bands were randomly selected for TA cloning.6 clones were selected for each sample.The methylation rate was calculated according to the actual percentage of the CpG loci in the total TA clones.7 DNA sequencing of the amplified products of gastric cancer cell lines was performed by traditional PCR to detect the mutation of IκBα gene.8 Predicting that the EBV-encoded miR-BART16 contains the binding site of IκBα 3’UTR by using the website of miRBase and rna22,the target sequence of IκBα 3’UTR or its mutant sequence were cloned into the double luciferase reporter gene plasmid,and the reporter gene plasmid was co-transfected into HEK293 T cells to detect the expression of luciferase.9 The plasmid containing the latent membrane protein LMP1 of EBV was constructed and transfected into EBVnGC cell line SGC7901.The cells transfected for 2h,6h,24 h,48h,and 72 h were collected and the proteins were extracted.The changes of the related proteins were detected by Western blot.LMP1 siRNA and control(NC)were designed and synthesized,transfected into EBVaGC cell line GT38,collected for 48 hours,extracted protein and RNA,Western blot and qRT-PCR were used to detect the expression of related genes.10 The plasmid containing the latent membrane protein LMP2 A of EBV was constructed and transfected into EBVnGC cell line SGC7901.The cells transfected for 2h,6h,24 h,48h,and 72 h were collected and the proteins were extracted.The changes of the related proteins were detected by Western blot.11 LMP1,LMP2 A plasmids and control plasmid were transfected into EBVnGC cell line SGC7901 respectively.After G418 screening and flow cytometry,SGC7901 cell lines with stable expression of LMP1,LMP2 A and control plasmid were obtained.The expression of genes related to NF-κB signaling pathway was detected by Western blot.12 The small interfering sequence of TRAF1 was designed and synthesized and transfected into EBVaGC cell line GT39 to detect the expression of NF-κB signaling pathway-related proteins.13 The relative expression of miR-BART16 and LMP1 in EBVaGC cell lines GT38,GT39 and 10 EBV-positive gastric cancer tissues were detected by qRT-PCR and immunohistochemical staining.14 The mimics and inhibitors of miR-BART16 were transfected into GT38 and GT39 cell lines at concentrations of 10,30 and 50 nM respectively.After 48 hours,the cells were collected and the relative expression of LMP1 was detected by extracting RNA and total protein respectively.After 24,48 and 72 hours of transfection,CCK-8 was used to detect the effect of miR-BART16 on cell proliferation.15 The effect of miR-BART16 on apoptosis was detected by flow cytometry with annexin V and PI double staining.PI and BrdU staining were used to analyze the effect of miR-BART16 on cell cycle.Results: 1 The transcriptional expression of NFKBIA in EBV-positive gastric cancer cell lines was significantly lower than that in negative cell lines(P<0.005).The transcriptional levels of TRAF1 and TRAF3 in EBV-positive gastric cancer cell lines were higher than those in negative gastric cancer cell lines,and TRAF2 were significantly lower than those in negative gastric cancer cell lines.There was no significant difference in the expression of p-IκBα and no significant difference in the expression of p65 and p-p65 in EBV-positive gastric cancer cell lines.The expression of TRAF1 in EBVaGC cell lines was significantly higher than that in negative cell lines.The protein expression of TRAF2,TRAF3 and TRAF6 in EBVaGC cell line was lower than that in negative cell line.2 The intranuclear translocation of positive gastric cancer cell lines accounted for about 50%,while that of negative cell lines was less or not detected.The p65-DNA binding ability of EBVaGC cell lines GT38,GT39 and SNU719 was higher than that of negative cell lines.3 The mRNA expression of IκBα in EBVaGC tissue was significantly lower than that in EBVnGC tissue and adjacent tissues.The expression of IκBα in EBVaGC tissue was weaker than that in EBVnGC tissue,while the expression of P65 was stronger than that in negative tissue,and the number of translocated tumor cells was more than that in negative tissues.4 The sensitivity of EBV-positive gastric cancer cell line SNU719 to the inhibitor of NF-κB signaling pathway BAY11-7082 was significantly stronger than that of EBV-negative gastric cancer cell line SGC7901.5 Compared with DMSO control group,the apoptotic rate of BAY11-7082 treatment group with 2.5μM concentration had no significant change(P>0.5).The apoptotic rate of BAY11-7082 treatment group with 5μM and 10μM concentration was significantly increased,the highest was 51.5%.There was a significant difference between BAY11-7082 treatment group and control group(P<0.001).Both MG-132 and BAY11-7082 signaling pathway inhibitors can affect the expression of apoptosis-related proteins in different degrees.6 There was no significant difference in ZEB1 protein expression(P>0.5)with BAY11-7082 treatment at 2.5μM concentration,but the expression of ZEB1 protein was significantly decreased at 5μM and 10μM concentrations(P< 0.01).7 The methylation rates of NFKBIA promoter region EBVaGC cell lines were GT38(0.80%),GT39(0.80%)and SNU719(0.4%)respectively,which were all hypomethylated.8 The deletion of 12-bp fragment(+615~+626 delCGCCCAGGAG)in exon 1 of EBVaGC cell lines GT38,GT39 and SNU719 resulted in the loss of 4 amino acids,which was not detected in EBVnGC cell lines.9 EBV-miR-BART16 could not directly regulate the expression of luciferase with h-NFKBIA-3’UTR.10 Compared with the blank control group,the expression of IκBα in the experimental group transfected with LMP1 was down-regulated in all time periods.The expression level of p-IκBα was 40% at 72 h than that in the control group.The highest expression of p-IκBα was reached at 7h(2.15 folds).The expression of total p65 protein was lower than that in the control group,while the expression of p-p65 was up-regulated.The expression of TRAF1 was up-regulated,reaching 2.01 times at 72 h,while the expression of TRAF2/3/6 was down-regulated.The expression of LMP1 was 26% of the control group,but the expression of IκBa did not change significantly(P>0.05).The expression of LMP1 protein was down-regulated,and the expression of IκBα protein was up-regulated.11 Compared with the blank control group,the expression of IκBα in the experimental group transfected with LMP2 A was down-regulated in all time periods,the most obvious decrease was at 48 h,the expression level was only 29% of the control group,and the expression of p-IκBα was up-regulated,reaching the highest level at 72h(3.32 times).The expression of total p65 protein was lower than that of the control group,while the expression of p-p65 was up-regulated sequentially.The expression of TRAF1 was up-regulated,reaching 2.51 times at 72 h,while the expression of TRAF2/3 was down-regulated,TRAF6 was down-regulated slightly.12 Compared with blank control,stable transfection of LMP1/2A could down-regulate the expression of IκBα,which was only 29% of control group.The expression of p-IκBα was up-regulated and reached the highest level(3.32 times)at 72 hours.The expression of total p65 protein was lower than that of control group,while the expression of p-p65 was up-regulated sequentially.13 The total protein and phosphorylated protein of p65 and IκBα did not change significantly after 48 h and 72 h transfection of the small interfering sequence of TRAF1 into EBVaGC cell line GT39.14 Compared with the GT38 and GT39,the expression of miR-BART16 in gastric cancer tissue was significantly higher,about 14-165 times.The expression of LMP1 was detected by qRT-PCR and immunohistochemistry in cell lines and gastric cancer tissues.No expression of LMP1 was detected in 10 gastric cancer tissues.15 Compared with negative control,the levels of RNA and protein expression in GT38 and GT39 cell lines transfected with the mimic of miR-BART16 decreased with the increase of concentration,while the levels of RNA and protein expression in GT38 and GT39 cell lines transfected with the inhibitor of miR-BART16 increased with the increase of concentration,showing a significant quantitative effect.16 The proliferation of cells was significantly inhibited at 48 h and 72 h after mimics transfection(P<0.05),while the proliferation was significantly increased after transfection(P<0.05).17 The apoptosis of GT38 and GT39 cells transfected with mimic and inhibitor of miR-BART16 did not change significantly(P>0.05).After 48 hours of transfection with the inhibitor of miR-BART16,compared with the control group,the cell cycle arrest was at G2/M phase,but there was no significant change after transfection of the mimics.Conclusion: 1 Both EBV-positive gastric cancer cell lines and EBVaGC tissues have sustained activation of the classical signal pathway of NF-κB.The expression of LMP1 and LMP2 A is an important reason for the activation of this signal pathway.2 The activation of NF-κB signaling pathway can promote the expression of epithelial mesenchymal transition-related molecules and latent membrane proteins,to promote the proliferation of EBV-related tumors,inhibit apoptosis.3 A 12 bp deletion in exon 1 of IκBα gene in EBVaGC cell line resulted in the loss of four amino acids.4 The low expression of IκBα gene in EBVaGC is not regulated by promoter methylation and miR-BART16.5 miR-BART16 can regulate the expression of LMP1 at the level of mRNA and protein,inhibiting the expression of miR-BART16 could promote cell proliferation and the ratio of G2/M phase cells. |
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