| Objective:This study aimed to find the WNT5A epigenetic mechanism and its effect on the development of EBVaGC. To understand the promoter methylation status and the expression at the protein level of retinoblastoma (Rb) and transforming growth factor-β1(TGF-β1) gene in Epstein-Barr virus (EBV)-associated and-negative gastric carcinomas for identifying the correlation of EBV with methylation and expression of Rb and TGF-β1.Methods:①20gastric carcinoma cell lines (5EBV+and15EBV-) were analyzed for their expression of WNT5A by RT-PCR.②The promoter methylation of WNT5A was detected by methylation-specific PCR and bisulfite genomic sequencing in these gastric carcinoma cell lines and clinical tissue samples of23EBVaGC,25EBV negative gastric carcinoma (EBVnGC), and their matched adjacent normal tissues.③Quantitative Real-Time PCR was used to detecte the expression of WNT5A expression after demethylated reagent treatment.④After transfection with pcDNA3.1-WNT5A Western Blotting or RT-PCR was used to access the protein expression of β-Catentin c-Jun and JunB and RNA expression of ATF2, c-myc and EGFR in EBV positive cell lines (SNU719).⑤Tirty EBV-associated gastric carcinoma (EBVaGC) and38matched EBV-negative gastric carcinoma (EBVnGC) tissues were examined for the promoter methylation of Rb and TGF-β1by methylation-specific PCR (MSP).⑥The expression of Rb and TGF-β1in gastric carcinoma tissues was detected by immunohistochemistry.⑦The mRNA expression of MMP9, Survivin, CDK4, EGFR and ICAM-1were detected by RT-PCR in EBV-positive and-negative cell lines after treatment with siRNA-LMPl or/and TGF-β1.Results:①WNT5A was common expression in EBV negative cell lines,, while loss of expression or downregulation in EBVpositive cell lines by RT-PCR.②WNT5A promoter was hypermethylated or partial methylated in all EBV positive cell lines, while it was partial methylated in some EBV negative cell lines by MSP. WNT5A methylation rate EBV positive cell ines was obvious higher than that in EBV negative cell lines by BGS(F=305.414,P<0.05).③There was significant difference of WNT5A promoter methylation between23pairs of EBVaGC and25pairs of EBVnGC tumors by using two independent sets of MSP primers (WNT5Am1/m2:χ2=7.561, P=0.006; WNT5Am3/m4:χ2=5.298, P=0.0214). The WNT5A promoter methylation was compared between EBVaGC tumors, EBVnGC tumors and their matched adjacent normal gastric tissue. There was obvious difference between EBVaGC tumors and their matched adjacent normal gastric tissue(WNT5Aml/m2:x2=18.050, P=0.000; WNT5Am3/m4:x2=10.083, P=0.0015), while there wasn't difference between EBVnGC tumors and their matched adjacent normal gastric tissue (WNT5Aml/m2: X2=3.500, P=0.061; WNT5Am3/m4:x2=2.286, P=0.131).④Demethylation reagents could restore WNT5A expression in these cell lines along with their demethylation.⑤Increased expression of WNT5A in vitro inhibited (3-Catentin expression.⑥The protein expression of c-Jun and c-myc were not different, JunB was increased, the mRNA expression of ATF2and EGFR were decreased expression wasn't different in SNU719after pcDNA3.1-WNT5A transfected compared with plasmid control, there was statistics significant (P<0.05).⑦The methylation rate of Rb gene in EBVaGC and EBVnGC was80.0%(24/30) and50.0%(19/38), respectively, with statistically significant difference (x2=6.490, P=0.011).The methylation rate of TGF-β1gene in EBVaGC and EBVnGC was (50.0%,15/30) and (36.8%,14/38), respectively, without statistically significant difference (x2=1.187, P=0.276).⑧There was no statistically significant difference for Rb expression between EBVaGC (43.3%,13/30) and EBVnGC (63.2%,24/38),as well as for TGF-β1expression between EBVaGC (56.7%,17/30) and EBVnGC (63.2%,24/38). Rb and TGF-β1methylation was not reversely correlated with Rb and TGF-β1expression in gastric carcinoma tissues (x2=2.943,P=0.086, r=0.208),(x2=3.051, P=0.081, r=0.212). Rb methylation was significantly associated with invasion depth and lymph node metastasis (P<0.05), but not was associated with gender,age,histological subtype (differentiation status) and tumor location. TGF-β1methylation was significantly associated with invasion depth and tumor location, but not was associated with gender, age, histological subtype (differentiation status) and lymph node metastasis (P<.05).⑨After treatment with siRNA-LMPl, siRNA-LMP1+TGF-β1, the expression of MMP9,Survivin and CDK4was decreased in EBV positive cell line (GT38) and CDK4was increased, the difference was significant (P<0.05); After siRNA-LMPl+TGF-β1, the expression of EGFR was increased compared with the normal control, while there wasn't obvious difference between siRNA-LMP1and normal control.⑩Compared with the normal control, the expression of ICAM-1was decreased in GT38,not in other cell lines after TGF-β1treatment in EBV positive cell lines (GT38and SNU719) and EBV negative cell lines (SGC7901and HGC-27)(P<0.05). In four cell lines the mRNA expression of MMP9, Survivin, CDK4and EGFR wasn't different between before and after TGF-β1treatment.Conclusion:①WNT5A expression is more common in EBV negative cell lines,while WNT5A was lost expression or downregulation in EBV positive cell lines.Demethylated reagent can restore the expression of WNT5A.The WNT5A promoter hypermethylation is the important mechanism of WNT5A silence or downregulation in EBV positive cell lines.②WNT5A promoter hypermethylation in EBVaGC tumors was obvious higher than that in EBVnGC tumors, indicating that WNT5Ais involved with the development of EBVaGC, which is the important epigenetic mark.③Ectopic introduction of WNT5A exhibits tumor-suppressive activity by takeing an effect on β-Catenin, JunB, ATF2and EGFR, indicating that WNT5A plays a negative regulation in the development of EBVaGC, which may have preventive/therapeutic potentials for the tumors with silenced WNT5A.④Methylation of Rb is a common event in gastric carcinomas and hypermethylation of Rb induced by EBV may contribute to the development of EBVaGC. EBV infection in gastric carcinoma tissues doesn't take any effect on TGF-β1promoter methylation and protein expression.⑤Rb methylation, loss of Rb expression and TGF-β1expression may be the reference indexes for judging the depth of invasion and lymph node metastasis of gastric carcinomas.⑥LMP1could play a key role in the expression of MMP9, Survivin and CDK4by LMP1-associated signal pathway. The expression of ICAM-1and EGFR are regulated by the interaction of LMP1and TGF-β1.
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