Expression of Dnmt1, demethylase, MeCP2 and methylation of tumor-related genes in human gastric cancer

Abstract

Aim: To explore the effect of DNA methyltransferase, demethylase and methyl-CpG binding protein MeCP2 on the expressions and methylation of hMSH2 and proto-oncogene in human gastric cancer.

Methods: Paired samples of primary gastric cancer and corresponding para-cancerous, non-cancerous gastric mucosae were obtained from surgically resected specimens of 28 patients. Transcription levels of Dnmt1, mbd2, MeCP2, p16(INK4A), hMSH2 and c-myc were detected by using real-time PCR or RT-PCR. Promoter methylation of p16(INK4A), c-myc and hMSH2 genes was assayed by methylation-specific PCR (MSP) and sequencing (mapping). Their relationships were analyzed by Fisher's exact test using the software SPSS.

Results: The average mRNA level of Dnmt1 gene from cancerous tissue was higher and that of mbd2 gene from cancerous tissue was lower than that from non-cancerous tissue, respectively. mbd2 was lower in cancerous tissue than in non-cancerous tissue in 14 (50.0%) of patients but higher in 3 cases (10.7%) of non-cancerous gastric tissue (P<0.001). c-myc expression was up-regulated in cancer tissues (P<0.05). The up-regulation of mbd2 was found in all patients with hypomethylated c-myc. The transcriptional levels of p16(INK4A) and MeCP2 genes did not display any difference between gastric cancerous and matched non-cancerous tissues. There were down-regulation and hypermethylation of hMSH2 in cancer tissues, and the hypermethylation of hMSH2 coexisted with down-regulated transcription. However, the transcription level of the above genes was not associated with biological behaviours of gastric cancers.

Conclusion: The up-regulation of proto-oncogene may be the consequence of epigenetic control of gene expression by demethylase, and mbd2 is involved in the regulation of hMSH2 expression in human gastric cancer.

Figure 1

mRNA expression of p16^INK4A, c-myc, mbd2 and MeCP2 in cancerous (lanes 1, 4, 7 and 10), para-cancerous (lanes 2, 5, 8 and 11) and non-cancerous (lanes 3, 6, 9 and 12) tissues from patients with gastric cancer by using RT-PCR.

Figure 2

Density of bands from RT-PCR in each lane normalized to the total RNA as determined by the density of bands in RT-PCR for β -actin. Assuming β -actin was 4 562.39 units (pixels of brightness), the calculation [4 562.39/(density of actin)] × (density of each gene) equals each gene normalized to β -actin. Data shown are representative of three separate experiments. ^aP < 0.05.

Figure 3

Hypermethylation of p16^INK4A promoter (A) and hypomethylation of proto-oncogene c-myc promoter (B) in gastric cancer tissue indicated by MSP. U: unmethylation-spe-cific PCR; M: methylation-specific PCR.

Figure 4

Methylation analysis of hMSH2 gene promoter by bisulfite sequencing. The arrows indicate the changes of CG to TG in promoters of non-cancerous and para-cancerous tissue, but CG remained in cancerous tissues.