Gastric Cancer

, Volume 21, Issue 4, pp 606–616 | Cite as

Clinical and biological significance of a − 73A > C variation in the CDH1 promoter of patients with sporadic gastric carcinoma

Original Article

Abstract

Background

CDH1 germline mutations lead to hereditary diffuse gastric carcinomas. However, it is unclear whether genetic variations in the CDH1 promoter affect the progression of sporadic gastric carcinomas (SGCs).

Methods

SGC patients in two independent cohorts with follow-up data were enrolled. The CDH1 genotypes, including the − 73A > C polymorphism (rs28372783), were determined by PCR sequencing. The CDH1 promoter activity was determined using reporter assays. SNAIL bound to CDH1 alleles was determined by chromatin immunoprecipitation primer extension PCR. CDH1 DNA methylation was determined by bisulfite-based PCR analyses.

Results

Kaplan–Meier analyses showed that the overall survival (OS) of the − 73C/C patients was significantly longer than that of the − 73A/C or − 73A/A patients in a Chinese cohort [n = 526; hazard ratio 0.68 (95% CI 0.47–1.00)], which was validated in an independent Korea cohort [n = 215; hazard ratio 0.49 (95% CI 0.26–0.94)]. Moreover, the transcription activity of the − 73C alleles was significantly higher than that of the − 73A alleles in vitro and in vivo. The ratio of SNAIL recruited to the promoter regions of the − 73C and − 73A alleles was 1:10, indicating a strong influence of this polymorphism on the recruitment of SNAIL to the flanking E-box. The prevalence of DNA methylation of the CpG island and shore within the promoter of the − 73C allele was much less than that of the − 73A allele in both gastric tissues and cancer cell lines.

Conclusion

The − 73A > C variation may lead to differences in the overall survival of SGC patients and allele-specific repressions of CDH1.

Keywords

CDH1 SNP Gastric carcinoma Overall survival Allele-specific repression DNA methylation SNAIL 

Notes

Acknowledgements

This work is supported by funding from the National Natural Science Foundation of China (nos. 81572762 and 31261140372) and the National Basic Research Program of China (no. 2015CB553902) to B. Zhang, WH Kim, and D. Deng. We thank Dr. Huidong Shi at the GRU Cancer Center at Georgia Reagents University, Augusta (USA) for language editing.

Compliance with ethical standards

Conflict of interest

The authors disclose no potential conflicts of interest.

Ethical approval

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions.

Informed consent

Informed consent or a substitute for it was obtained from all patients before they were included in the study unless the local institution review board permitted a waiver.

Supplementary material

10120_2017_778_MOESM1_ESM.tif (149 kb)
Locations of the SNPs, CpG sites, and various PCR amplicons within the CDH1 CpG islands, 5’-shore, and the Alu element. TSS transcription start site (+1-nt), E-boxes; SNAIL binding sites (TIFF 149 kb)
10120_2017_778_MOESM2_ESM.tif (246 kb)
Comparison of the promoter activity of the –73A allele with that of the –73C allele in various human cancer cell lines. a The methylation status of the CDH1 promoter detected by the 115-bp methylation-specific PCR. b and c Levels of protein and mRNA of CDH1 detected by western blot and RT-PCR, respectively; cDNA from HeLa cells was used as the CDH1-expression-negative control. d Comparison of the promoter activity of the –73C allele with the –73A allele in various cell lines. * p<0.001 (TIFF 245 kb)
10120_2017_778_MOESM3_ESM.tif (223 kb)
Graphic view of the methylation status of the CpG island and the 5′-shore regions around the transcription start site of the CDH1 gene in various human cell lines. The methylation datasets were detected using reduced representative bisulfite sequencing. This image was modified from the image for the CDH1 gene visualized with the UCSC Genome Browser (Human hg19) found at the web site http://genome.ucsc.edu. Each red bar represents a methylated CpG site; the yellow bar represents a partially methylated CpG site; the green bar represents a CpG site that is not methylated. (TIFF 222 kb)
10120_2017_778_MOESM4_ESM.tif (504 kb)
Graphical view of the methylation status of the CpG island and shore regions around the transcription start site of the CDH1 gene in various adult human tissues/cells. The methylation datasets were detected using human whole genome bisulfite sequencing. This image was modified from the image for the CDH1 gene visualized with the WashU EpiGenome Browser (Human hg19) found at the web site http://epigenomegateway.wustl.edu/browser/. The height of each blue bar represents the combined read depth for the corresponding methylated CpG site; the height of each gray bar represents the combined read depth of the corresponding CpG. (TIFF 504 kb)
10120_2017_778_MOESM5_ESM.tif (2.1 mb)
Comparison of the methylation statuses of the CpG sites in the promoter CpG island and the shore regions in the representative gastric tissue sample #0187N from a patient carrying the –347del/del and –160C/C genotype of CDH1. The proportion of the –73A clones that were underlined clones containing at least three methylated CpGs in the shore region was much higher than the corresponding proportion of the –73C clones [12/129 vs. 1/70 (p=0.036)]. Red dots represent methylated CpG sites. (TIFF 2122 kb)
10120_2017_778_MOESM6_ESM.tif (2.1 mb)
Comparison of the methylation statuses of the CpG sites in the promoter CpG island and the shore regions in the representative gastric tissue sample #0379N. The proportion of the –73A clones that were underlined clones containing at least three methylated CpGs in the shore region was much higher than the corresponding proportion of the –73C clones [12/145 vs. 1/52 (p=0.190)]; the proportions were 2/21 vs. 11/176 in the −347A and –347del clones. Red dots represent methylated CpG sites. (TIFF 2117 kb)
10120_2017_778_MOESM7_ESM.docx (17 kb)
Supplementary material 7 (DOCX 16 kb)

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Copyright information

© The International Gastric Cancer Association and The Japanese Gastric Cancer Association 2017

Authors and Affiliations

  1. 1.Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of EtiologyPeking University Cancer Hospital and InstituteBeijingChina
  2. 2.Department of SurgeryPeking University Cancer Hospital and InstituteBeijingChina
  3. 3.Department of PathologySeoul National University College of MedicineSeoulKorea

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