Role Of DNA Methylation Of FES And CLDN11 Gene In Hepatocellular Carcinoma

Purpose:To search for new methylation biomarkers of hepatocellular carcinoma (HCC), and to further explor the epigenetic mechanisms of their transcriptional regulation.Design:(1) The global methylation profile of HCC was analyzed using Illumina 450K Infinium Methylation BeadChip.(2) The methylation status of six genes (FES> CLDN11, CYP26C1, FBN1、 SEPT9, PITX2) in 100 HCC and 100 matched noncancerous specimens tissues were examined using methylation-specific PCR (MSP).(3) The methylation status of FES and CLDN11 in 30 HCC and 30 matched noncancerous specimens tissues were examined using pyrosequencing.(4) The expression level of FES and CLDN11 were determined by immunohistochemistry (1HC).(5) The association between the methylation status and expression level of FES and CLDN11 with clinicopathological parameters were also analyzed. The survival curves were plotted using Kaplan-Meier method and the differences in overall(OS)or progression-free survival(PFS) were assessed with the log-rank test. Univariate and multivariate survival data was analyzed using a Cox regression model, which was used to identify whether FES and CLDN11 could be the independent prognostic factor.(6)The human hepatocellular carcinoma cell line BEL-7402 were treated with 5-Aza-CdR and TSA either alone or in combination.Changes of methylation status, mRNA expression and protein expression of FES and CLDN11 in each group of cells were investigated by MSP,qRT-PCR and western blot respectively.Results:(1) Aberrant methylated genes were enriched in pathways including RNA transport, Oxidative phosphorylation, Cell cycle, Cytokine-cytokine receptor interaction and Metabolic pathways. We selected 6 genes(FES、 CLDN11、 CYP26C1、FBN1、 SEPT9、 PITX2) for future validation.(2) The FES and CLDN11 methylaiton were more frequent in HCC tissues than noncancerous specimens tissues by MSP.(3) The FES and CLDN11 methylaiton were more frequent in HCC tissues than noncancerous specimens tissues by pyrosequencing.(4) The expression of FES and CLDN11 was down regulated in HCC tissues compared with matched noncancerous specimens tissues.(5) The results demonstrate that the FES methylation was associated with tumor differentiation, AFP level and tumor size. The expression level of FES was associated with tumor differentiation, AFP level and tumor size. The CLDN11 methylation was associated with tumor differentiation and tumor size. The expression level of CLDN11 was associated with tumor differentiation, tumor size and Tumor metastasis. Kaplan-Meier survival analysis show that patients with tumor differentiation, AFP level, tumor size, hypermethylation of FES and CLDN11, expression level of FES and CLDN11 correlate were associated with patients’ OS and PFS. Univariate and multivariate survival data demonstrated that AFP level, tumor size, tumor differentiation and tumor metastasis as the independent prognostic factor for patients with HCC.(6) Both of FES and CLDN11 complete methylation were detected in untreated 7402 cells and were partially reversed by 5-Aza-CdR or TSA, mRNA and protein expression was increased and TSA was more efficient than 5-Aza-CdR in inducing gene expression. After treatment by the combination of the two inhibitors, FES and CLDNll methylation was completely reversed and mRNA and protein expression was significantly increased.Conclusions:The promoter of FES and CLDNll were hypermethylation in hepatocellular carcinoma, and the proteins were down-regulation. After treatment by 5-Aza-CdR or/and TSA, FES and CLDNl 1 methylation was completely reversed and mRNA and protein expression was significantly increased.