EBV Down-regulates COX-2 Expression Via TRAF2 And ERK Signal Pathway In EBV-associated Gastric Cancer

Objective:Gastric malignant tumors are characterized by high morbidity and high mortality,and gastric cancer associated with Epstein-Barr virus(EBV),namely EBV-associated gastric cancer(EBVaGC),accounts for approximately 10%,so it is of great significance to study the mechanism of EBVaGC and its related genes.However,there has been no report of the correlation between EBV-encoded Latent Membrane Protein 2A(LMP2A)and cyclooxygenase-2(COX-2)in EBV-associated tumors.Whether EBV-encoded Latent Membrane Protein 1(LMP1)interacts with COX-2 in EBVaGC also needs further study.Methods:(1)3 EBV positive gastric cancer(EBVaGC)cell lines(GT38,GT39 and SNU719)and 5 EBV negative gastric cancer(EBVnGC)cell lines(SGC7901,BGC823,AGS,HGC27 and MKN45)were selected and used to detect whether there is a difference in the expression level of COX-2 by real-time fluorescent quantitative PCR(Quantitative Real-time PCR,qRT-PCR)method.(2)Bisulfite Genomic Sequense(BSP)was used to defind the methylation level of the COX-2 gene promoter region in EBV associated/negative gastric cancer cell lines.(3)qRT-PCR method to test the transcription expression of several microRNAs targeting COX-2 encoding gene.(4)Western Blot method was used to discover the protein expression levels of COX-2 and TRAF2(Tumor necrosis factor receptor-related factor 2)in EBV positive/negative gastric cancer cell lines.(5)In the EBVnGC cell line SGC7901,pcDNA3.1 was used to construct recombinant plasmids encoding LMP1/2A,and the effect of LMP1/2A on the expression of COX-2and TRAF2 was observed.(6)In the EBVaGC cell line GT38,small interfere sequences(siRNA)were used to target LMP1/2A,respectively,to observe the effects of its expression silencing on COX-2 and TRAF2.(7)In SGC7901,two different sequences of si-TRAF2 were used to ensure the accuracy of its regulation of COX-2 expression by silencing TRAF2.(8)The MEK inhibitor PD0325901 and si-ERK1/2 were used to block the ERK pathway in SGC7901 to detect whether p-ERK is involved in the regulation of COX-2 by LMP1.(9)EBV can activate PI3K/AKT pathway,use PI3 K inhibitor LY294002 to block AKT pathway in GT38,and study the role of p-AKT in the regulation of COX-2 by EBV.(10)si-FoxO1 was used to interfere with the expression of FoxO1 inSGC7901,and the mRNA and protein levels of COX-2 were detected.Results:(1)The mRNA expression of COX-2 in the three EBVaGC cell lines was lower than that of the five EBVnGC cell lines(t=10.3,P=0.0005).(2)The methylation level of the COX-2 gene promoter region in EBVaGC cell line and EBVnGC cell line was 4.12%and 11.42%,respectively.Statistical analysis showed that the methylation level of the COX-2 gene promoter region was significantly lower in the EBVaGC cell line than in the EBVnGC cell line(t=2.58,P=0.04).(3)The COX-2 protein levels in the EBVaGC cell line and the EBVnGC cell line were significantly different.Statistical analysis showed that the COX-2 protein level in the EBVaGC cell line was significantly lower than that of the EBVnGC cell line(t=6.075,P=0.0017).(4)The exogenous transfection of LMP1 or LMP2 A plasmid in EBVnGC cell line SGC7901 significantly reduced the expression of COX-2 and TRAF2;interference with LMP1 or LMP2 A in GT38 of EBVaGC cells restored the expression of COX-2 and TRAF2 protein.(5)After interference with TRAF2 in SGC7901,a sharp decrease in COX-2 was observed.(6)In addition,overexpression of LMP1 in AGS reduced ERK activation(t=7.49,P=0.01).Using MEK inhibitors and si-ERK1/2 to identify ERK blockade down-regulated COX-2 protein expression(P<0.01).(7)After using PI3 K inhibitor LY294002 in GT38,it was found that the downstream transcription factors Fox O1 and COX-2 were restored,and the inhibition of Fox O1 in SGC7901 reduced the production of COX-2(t=3.68,P=0.02),Therefore it was identified that FoxO1 is involved in the inhibition of COX-2 by p-AKT.Conclusion:(1)In gastric cancer cells,overexpression of LMP1 and LMP2 A can inhibit COX-2 expression by down-regulating TRAF2.(2)p-ERK is involved in the inhibitory effect of LMP1 on COX-2.(3)It was observed that EBVaGC cells can reduce the transcription of COX-2 by activating the AKT pathway to phosphorylate FoxO1.