| Background and objective: Epstein-Barr virus(EBV)is involved in the carcinogenesis of EBV-associated tumors through interfering the gene expression of host cells.As the hub of multiple signaling pathways,caveolin-1(CAV-1)plays a vital role in the occurrence and the development of various tumors.CAV-1 has been shown to play roles both as a tumor promoter or a tumor suppressor,which may depend on the different tumor tissue and cell types.The role of CAV-1 in gastric carcinoma remains controversial.Due to the existence of EBV,EBV-associated gastric carcinoma(EBVaGC)has unique clinicopathological and molecular characteristics,forming a special subtype of gastric carcinoma.The data from microarray and our previous result indicated that EBV may inhibit the expression of CAV-1 in gastric carcinoma cells.This study aims to determine the effect of EBV on CAV-1 expression and to investigate the mechanism of EBV regulating CAV-1 expression in gastric carcinoma,and also explore the role of CAV-1 in the progression of gastric carcinoma.It would provide evidence for the carcinogenic mechanisms of EBV.Methods: The expression of CAV-1 in EBV-negative and-positive gastric carcinoma cell lines was detected by RT-qPCR and Western blotting,and the expression CAV-1 in EBVaGC and EBV-negative gastric carcinoma(EBVnGC)tissues and para-carcinoma tissues was detected by immunohistochemistry.Targeting inhibition of EBV-mir-BART1-3p on CAV-1 was detected by miRNA mimics and inhibitor transfection.The methylation of CAV-1 in gastric carcinoma cell lines and in gastric carcinoma tissues was detected by bisufite genomic sequencing(BGS)and methylation-specific PCR(MSP),respectively.The expression of DNA methyltransferases(DNMTs),including DNMT1,DNMT3 a and DNMT3 b,in gastric carcinoma cell lines,and the expression of CAV-1 in EBV-positive cell lines which was treated by 5-Aza-dC were detected by RT-qPCR.The biological behavior of gastric carcinoma cells stably transfected with CAV-1 siRNA lentivirus was detected by CCK-8,plate clone formation assay,apoptosis induction assay and migration and invasion assay.Results:(1)The transcriptional and protein level of CAV-1 in EBV-positive gastric and nasopharyngeal carcinoma cell lines were significantly lower than those in EBV-negative gastric and nasopharyngeal carcinoma cell lines(P<0.05),and also the positive expression rate of CAV-1 in EBVaGC tissues(2.5%,1/40)was significantly lower than that in EBVnGC tissues(22.6%,12/53)(P<0.05).(2)Three EBV encoding miRNAs were predicted to bind with the 3'UTR of CAV-1 gene(P<0.05),of which the EBV-miR-BART1-3p was highly expressed in EBV-positive gastric carcinoma cells.However,the inhibitor and mimics of EBV-miR-BART1-3p had no significant effect on the expression of CAV-1 in EBV-positive and-negative cell lines.(3)The 37 methylation sites in the CAV-1 promoter and the first exon region were hypermethylated in the EBV-positive gastric carcinoma cell line,while those were hypomethylated in the EBV-negative gastric cancer cell lines(P<0.05).MSP results showed that 13 cases of 15 EBVaGC tissue specimens(86.7%,13/15)had methylation bands,and 12 cases of 20 EBVnGC tissue specimens(60.0%,12/20)had methylation bands.(4)The expression level of DNMT3 a in EBV-positive gastric carcinoma cell lines was significantly higher than that in EBV-negative cell lines,and the expression level of CAV-1 was increased in 5-Aza-dC-induced EBV-positive gastric carcinoma cell lines.(5)CAV-1 siRNA transfected EBV-negative BGC823 cells effectively inhibited CAV-1 expression,increased cell proliferation rate,platelet formation rate,anti-cisplatin-induced apoptosis and cell migration and invasion abilities(P<0.05).The positive expression rate of CAV-1 in gastric carcinoma tissues(26.5%,13/49)was significantly lower than that in matched para-carcinoma tissues(83.7%,41/49)(P<0.05).The positive expression rate of CAV-1 in 91 EBVnGC tissue cases was 28.8%(25/91),and it showed higher positive rate in differentiated gastric carcinoma tissues than undifferentiated type(P<0.05),and also higher in cases without lymph node metastasis than case with lymph node metastasis(P<0.05).Conclusion:(1)EBV may inhibits the expression of CAV-1 in EBVaGC through inducing hypermethylation of CAV-1 promoter but not by EBV-encoding miRNAs.(2)The expression of CAV-1 is down-regulated in gastric carcinoma tissues compared with para-carcinoma tissues.Gene silencing of CAV-1 in gastric carcinoma cells promote cell proliferation,migration,invasion and enhance its anti-apoptotic ability.It is suggested that CAV-1 may act as a tumor suppressor in gastric carcinoma. |
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