Methylation of neutral endopeptidase 24.11 promoter in rat hepatocellular carcinoma

Abstract

Neutral endopeptidase 24.11 (NEP), a cell-surface enzyme expressed by epithelial cells that cleaves and inactivates biologically active small peptides, is downregulated in various cancers. NEP is encoded by a gene that contains a CpG island in the promoter region, whose hypermethylation appears related to decreased expression. Altered expression of NEP has also been reported in human hepatocellular carcinoma (HCC), suggesting its possible role in hepatocarcinogenesis. To elucidate the status of NEP in HCC, methylation in the promoter region of the gene that encodes NEP in male Fischer 344 rats with HCC, induced by a choline-deficient, l-amino acid-defined diet, was investigated by methylation-specific polymerase chain reaction, combined bisulfite restriction analysis, and bisulfite genomic sequencing. These analyses together showed the promoter to be frequently methylated in HCC in contrast to its unmethylated status in normal liver, the degree of methylation being inversely related to the level of mRNA expression evaluated by reverse transcription-polymerase chain reaction (P = 0.031). In two rat liver cell lines, RLC-16 and RLC-27, the promoter was heavily methylated and NEP mRNA expression was negative. However, administration of 5-aza-2'-deoxycytidine caused NEP expression, suggesting that methylation of CpG is a factor regulating transcriptional expression. Together with the data from microarray analyses performed previously using the same animal model, the current results suggest that reduced expression of NEP or other ectopeptidases could impact on molecules involved in signal-transducing systems, including G-protein coupled receptors, via modified turnover of extracellularly active small peptides.

Figure 1

(a) Genomic structure of the rat NEP gene. The open boxes represent the exons and the hatched box the CpG island (CGI). (b) Nucleotide sequence of the rat NEP gene. The sequence is numbered from the exon 2 transcription start site, indicated as +1. Sequences of representative primers are underlined. CpG present in the indicated sequence are in italic and those analyzed by methylation‐specific polymerase chain reaction (MSP), combined bisulfite restriction analysis (COBRA), or bisulfite genomic sequencing are numbered. HinfI recognition sites for methylated CpG are indicated as closed triangles.

Figure 2

Methylation status of the NEP gene was assayed by methylation‐specific polymerase chain reaction (MSP), combined bisulfite restriction analysis (COBRA), and bisulfite genomic sequencing. (a) Examples of MSP analysis of hepatocellular carcinoma (HCC) and normal liver samples from control rats. Bisulfite‐modified genomic DNA from HCC (T) and normal control livers (N) was amplified with methylated DNA‐specific primers or unmethylated DNA‐specific primers. 16, RLC‐16; 27, RLC‐27. (b) Examples of COBRA with HinfI digestion. Polymerase chain reaction products from bisulfite‐treated DNA obtained from HCC (T) and normal livers (N) were treated with restriction endonuclease HinfI. Only specimens that had methylated CpG were restricted. The gels were stained with ethidium bromide. Unmethylated (top rows) and methylated (bottom rows, arrows) products were quantitated by densitometry. Methylation densities (percentage of restricted vs unrestricted fragments) are shown below each lane. (c) Results of bisulfite genomic sequencing. Normal liver tissue (N1), three HCC samples (T3, T7 and T9) and a rat liver cell line (RLC‐27) were analyzed. For each sample, eight clones for the region in the CpG island (Fig. 1) were sequenced. Each square indicates a CpG site and each line of squares represents the analysis of a single‐cloned allele. Methylated CpG sites are shown as closed squares and their unmethylated counterparts as open squares. (d) MSP analysis of surrounding, non‐cancerous tissues (NC). Bisulfite‐treated DNA samples NC1, NC3 and NC9 were obtained from rats that had HCC T1, T3 and T9, respectively. In NC3 and NC9, methylated primers also provided positive bands; however, by comparing the intensities of bands, the extent of methylation was considered weaker in surrounding areas than in corresponding HCC themelves (a).

Figure 3

(a) Examples of mRNA expression for NEP (upper panel) and β‐actin (lower panel) in hepatocellular carcinoma (HCC) and normal liver tissues from control rats assayed by semiquantitative reverse transcription–polymerase chain reaction. (b) Expression of NEP at mRNA level in normal liver samples was greater than in HCC. Data are mean ± SD for ratios of NEP to β‐actin. The experiment for mRNA expression was performed in triplicate. *P < 0.05.

Figure 4

Increased expression of NEP transcripts in rat liver‐derived cells after exposure to 5‐aza‐2′‐deoxycytidine. The mRNA level of NEP expression in the cells was determined using semiquantitative reverse transcription–polymerase chain reaction before (–) and after (+) treatment with 10 µM 5‐aza‐2′‐deoxycytidine for 4 days. Top panel, NEP primer products; bottom panel, β‐actin primer products.