Study On Methylation Status And Expression Of RASSF1A Gene Promoter In EBV-associated Gastric Carcinoma

Objective: This study is to explore the methylation status of RASSF1 A gene in EBV positive and negative cell lines and gastric cancer tissues,and to further research the roles of RASSF1 A played in the occurrence of EBVaGC.Methods: Methylation-specific polymerase Chain Reaction(MSP)and Bisulfite genome sequencing(BGS)in combination with TA clone were used to detected methylation of RASSF1 A in 8 cancer cell lines(GT38,GT39,SNU719,AGS-EBV,HGC-27,MKN-45,SGC7901,AGS)and 77 gastric cancer tissues(36 EBVa GC and 41 EBVn GC).DNA-demethylating agent 5-Aza-2'deoxycytidine(5-Aza-dc)was used to demethylate the RASSF1 A promoter in 8 cell lines.RASSF1 A gene m RNA expression was detected by real time-PCR.The expression of RASSF1 A protein in gastric carcinoma tissues samples were investigated by immunohistochemistry(IHC).Results:(1)MSP results showed that RASSF1 A gene promoters were U type in four EBV positive gastric carcinoma cell lines(GT38,GT39,SNU719,AGS-EBV)and M type in four EBV negative gastric carcinoma cell lines(HGC-27,MKN-45,SGC7901,AGS).Detailed methylation analysis of each individual site in the RASSF1 A Cp G island by BGS.BGS results showed that only scattered methylated Cp G sites were detected in four EBV positive gastric carcinoma cell lines and densely methylated Cp G sites in the RASSF1 A promoter were found in four EBV negative gastric carcinoma cell lines.(2)MSP results showed that in 36 EBVa GC RASSF1 A gene promoters of 28 cases were M-type(28/36,77.8%)and 8 cases(8/36,22.2%)were M+U-type in.In 41 EBVn GC tissues,there were 33 cases were U-type(33/41,80.5%)and 8 cases(8/41,19.5%)were M+U-type.BGS results showed high methylation rate in 36 EBVa GC tissues.The mean methylation rate of RASSF1 A gene promoter in these 36 EBVa GC is 86%,while the mean methylation rate in EBVn GC is 22.3%.(3)After treatment with 5-Aza-dc,the methylation status changed to M+U type in four EBV negative cell lines,whereas no change was found in four EBV positive cell lines.(4)The transcriptional expression level of RASSF1 A gene in EBV positive gastric cancer cell line was higher than that in negative gastric cancer cell line.After 5-Aza-dc,the expression of RASSF1 A gene in EBV-negative gastric cancer cells was significantly increased,while EBV positive gastric cancer cell lines did not change.(5)There is no significant differences of RASSF1 A protein expression in EBVaGC and EBVnGC(P >0.05)Conclusion:(1)There is a reverse correlation between hypermathylation of RASSF1 A promoter and the presence of EBV in gastric cancer cell lines.(2)The promoter hypermethylation is correlated with RASSF1 A gene expression in EBV negative gastricl cancer cell lines,and plays an important role in RASSF1 A silencing.5-Aza-dc may effectively reverse the methylation status of RASSF1 A gene promoter in EBV negtive cell lines and reactivated gene expression silenced by aberrant hypermethylation.(3)The methylation status of RASSF1 A gene promoter region was different in EBVa GC and EBVn GC.EBVa GC was hypermethylated and EBVn GC was hypomethylated.These results indicating that EBV infection could induce the abnormal methylation of RASSF1 A and silence the gene.Thus,inactivation of the tumor suppressor gene might be a mechanism of EBV to participate the EBVa GC tumorigenesis.