Mechanism and pathobiologic implications of CHFR promoter methylation in gastric carcinoma

Abstract

Aim: To investigate the aberrant methylation of CHFR promoter in human gastric cancer (GC) and its impact on the expression of CHFR mRNA and protein, as well as its correlation with clinical and histological features of human GC.

Methods: Methylation-specific polymerase chain reaction (MSPCR) was used to detect the methylation status of CHFR promoter in 20 primary GC samples and paired normal gastric mucosa. The CHFR mRNA and protein expressions were investigated both by RT-PCR and by Western blotting. The CHFR protein expression in 39 GC samples was immunohistochemically examined.

Results: The DNA methylation of the CHFR gene was found in 9 of the 20 GC samples (45%) and the down-regulation of CHFR mRNA and protein was significantly associated with the methylation status of the CHFR gene (P = 0.006). In 20 samples of corresponding non-neoplastic mucosa, no DNA methylation of the CHFR gene was detected. The CHFR gene methylation in poorly differentiated GC samples was significantly higher than that in well-differentiated GC samples (P = 0.014). Moreover, the negative CHFR protein expression rate in paraffin-embedded GC samples was 55.07% (38/69), the positive rate in poorly differentiated GC samples was 36.73% (18/49), which was significantly lower than 65.00% (13/20) in well-differentiated GC samples (c2 = 4.586, P = 0.032).

Conclusion: Aberrant methylation of the CHFR gene may be involved in the carcinogenesis and development of GC, and is the predominant cause of down-regulation or loss of CHFR mRNA or protein expression. As aberrant methylation of CHFR promoter is correlated with tumor differentiation, it may help to predict the prognosis of GC and CHFR may become a novel target of gene therapy for GC in the future.

Figure 1

Representative results of MSPCR in human GC tissue samples. Lanes U and M: Products derived from unmethylated and methylated alleles, respectively. Methylation of the CHFR gene was detected in 16 and 6 poorly-differentiated adenocarcinoma tissue samples and 1 mucinous adenocarcinoma tissue sample, while unmethylation of the CHFR gene was detected in 7 well-differentiated adenocarcinoma tissue samples. PUC18: Marker; N: Normal gastric mucosa corresponding to tumors; T: Gastric cancer.

Figure 2

Representative results of RT-PCR in human GC tissue samples. β-actin was used as an internal control. CHFR mRNA expression level in 16 and 6 poorly differentiated adenocarcinoma tissue samples and 1 mucinous adenocarcinoma tissue sample was significantly lower than that in paired normal gastric mucosa samples from the same patients, while no difference was found in 7 well-differentiated adenocarcinoma tissue samples. Marker, D2000: Marker; N: Normal gastric mucosa corresponding to tumors; T: Gastric cancer.

Figure 3

Representative results of Western blot in human GC tissue samples. β-tubulin was used as an internal control. CHFR protein expression level in GS tissue samples was significantly lower than that in paired normal gastric mucosa samples. Aberrant methylation of CHFR and down-regulation or loss of CHFR mRNA expression were detected in 16 poorly differentiated adenocarcinoma tissue samples and 1 mucinous adenocarcinoma tissue sample, while positive protein expression of CHFR was detected in 9 moderately-differentiated adenocarcinoma tissue samples without CHFR methylation. N: Normal gastric mucosa corresponding to tumors; T: Gastric cancer.

Figure 4

Immunohistochemical staining for CHFR protein expression in GC tissue samples and normal gastric mucosa samples. Positive expression of CHFR in normal gastric mucosa tissue samples (A), in well-differentiated adenocarcinoma tissue samples (B), and in signet-ring cell carcinoma tissue samples (C).