The Role Of ET-1/ETAR Axis In Gastric Cancer And Its Relationship With EBV

Background: Epstein-Barr virus(EBV)is a double-stranded DNA virus that persists latent infection in host cells and is associated with a variety of human tumors.More than 90% of children aged 3 to 5 years have been infected with EBV,and more than 90% of adults can be detected EBV antibodies.EBV associated gastric carcinoma(EBVaGC)is a relatively high incidence of EBV-associated tumors,but its pathogenesis is not yet clear.ET-1 is a member of the endothelin family.Current studies have shown that ET-1 is elevated in most tumors and promotes tumorigenesis,cell migration and invasion,inhibition of apoptosis,and induction of epithelial-mesenchymal transition,playing an important role in the development process.ET-1 and its receptor ETAR are closely related to viral infections,such as HIV,HPV and HBV.FOXO1 is both a transcriptional activator of ET-1 and a downstream effector of the PI3K/AKT and RAS/RAF/MEK/ERK pathways.Altering the expression of FOXO1 can affect the expression of ET-1,which in turn affects the cascade reaction induced by ET-1/ETAR activation.Objective: To understand the role of ET-1/ETAR in gastric cancer and the effect of EBV on ET-1/ETAR expression.Materials and methods:(1)The expression of ET-1 axis and microRNAs in EBVaGC cell lines(GT38,GT39,SNU719)and EBVnGC cell lines(BGC823,HGC27,SGC7901,MKN45,AGS)by real-time quantitative PCR(RT-qPCR).(2)Exogenous ET-1 or ETAR-specific blocker BQ123 was added to the GT39 cell line to detect the effect of ET-1/ETAR on cells by CCK8,Transwell and apoptosis assays.(3)Bisulfite genomic sequencing(BGS)was used to detect the methylation status of ET-1 gene promoter and the first exon.(4)Western blotting was used to detect the expression of ET-1 and related genes in EBVaGC and EBVnGC cell lines.Immunohistochemistry was used to detect the expression of ET-1,ERK1/2 and p-ERK1/2 in EBVaGC and EBVnGC tissues.(5)The subcellular localization of ERK1/2 was detected by immunofluorescence test.(6)The expression of ET-1 protein in cells treated with PI3 K inhibitor LY294002 or MEK inhibitor PD0325901.(7)Overexpression of LMP1 or LMP2 A in the SGC7901 and its effect on ET-1 protein.(8)The changes of ET-1 expression after knocking down LMP1 or FOXO1.(9)ETAR-specific blocker BQ123 was added to GT39 and GT38,and different doses(0.01?M,0.1?M,1?M,10?M)and different time(0.5h,1h,3h,5h,7h,12h)were extracted.Changes in the expression of LMP1 protein.Results:(1)The mRNA and protein expression levels of ET-1 were lower in EBVaGC cell line than in EBVnGC cell line(P<0.05);RNA levels of ETAR genes,but not protein levels,are highly expressed in EBVaGC.(2)ET-1/ETAR activation promoted cell proliferation and cell migration and inhibited cisplatin-induced apoptosis.(3)Sequence of ET-1 promoter and first exon were both hypomethylated and the expression of miRNAs(-1,-125 a,-125b)was not significantly different in EBVaGC and EBVnGC.(4)Activation of PI3K/AKT pathway and inactivation of RAS/MEK/ERK pathway in EBVaGC could lead to decreased FOXO1 expression.(5)ERK1/2 was more active in EBVnGC paraffin sections(P<0.05),mainly distributed in cytoplasm and nucleus,and its expression was significantly correlated with age(P<0.05).(6)LY294002 could promote ET-1 expression by up-regulating FOXO1,while PD0325901 could down-regulate ET-1 protein levels by inhibiting FOXO1.(7)Knockdown of FOXO1 could down-regulate ET-1 protein level.(8)Knockdown of LMP1 could promote the expression of ET-1 and inhibit the expression of ETAR protein.(9)Overexpression of LMP1 gene in SGC7901 cells could inactivate Ras/MEK/ERK pathway and down-regulate ET-1 protein expression;while overexpression of LMP2 A gene could activate Ras/MEK/ERK pathway and up-regulate ET-1 expression.(10)ETAR-specific blocker BQ123 significantly inhibited the expression of LMP1 protein(P<0.05).Conclusion:(1)ET-1 was highly expressed in EBVaGC,and activation of ET-1/ETAR axis could promote cell proliferation,migration,and antagonize cisplatin-induced apoptosis.(2)Activation of the PI3K/AKT pathway in EBVaGC could inhibit the expression of FOXO1,leading to block the transcription of the ET-1 gene;the inactivation of Ras/MEK/ERK pathway in EBVaGC could reduce the expression of ET-1 by inhibiting the expression of FOXO1.(3)EBV regulated the expression of FOXO1 by altering the balance of PI3K/AKT pathway and Ras/MEK/ERK pathway,thereby affecting the expression of ET-1 This reseach studied the regulation mechanism of EBV on ET-1/ETAR axis and provided a new research direction for further study on the mechanism of EBV-positive gastric cancer and the formation of unique clinicopathological features.