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World J Gastroenterol. Aug 14, 2009; 15(30): 3799-3806
Published online Aug 14, 2009. doi: 10.3748/wjg.15.3799
Methylation of PTCH1a gene in a subset of gastric cancers
Peng Du, Hai-Rong Ye, Jun Gao, Wei Chen, Zhong-Chuan Wang, Hong-Hua Jiang, Ji Xu, Ji-We Zhang, Jian-Cheng Zhang, Long Cui
Peng Du, Wei Chen, Zhong-Chuan Wang, Hong-Hua Jiang, Ji Xu, Ji-We Zhang, Long Cui, Department of Surgery, Shanghai Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China
Hai-Rong Ye, Department of Anesthesiology, Shanghai Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China
Jun Gao, Department of Gastroenterology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
Jian-Cheng Zhang, Division of Ship Hygiene, Navy Medical Research Institute, Shanghai 200433, China
Author contributions: Du P and Ye HR performed the majority of the experiments; Ye HR and Xu J were involved in editing the manuscript; Chen W, Wang ZC, Jiang HH and Zhang JW collected all the human material; Zhang JC performed the methylation detection; Gao J and Cui L designed the study and wrote the manuscript; Du P, Ye HR and Gao J contributed equally to this work.
Correspondence to: Dr. Long Cui, Department of Surgery, Shanghai Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, 1665 Kong Jiang Road, Shanghai 200092, China. cuilongxh@yahoo.cn
Telephone: +86-21-65790000
Fax: +86-21-65790000
Received: May 15, 2009
Revised: July 2, 2009
Accepted: July 9, 2009
Published online: August 14, 2009
Abstract

AIM: To establish if PTCH1a transcriptional regulation region (TRR) is methylated in gastric cancer and its influence in gastric tumorigenesis.

METHODS: The CpG islands in PTCH1a TRR were analyzed by Methyl Primer Express v1.0 software. The region from -643 to -355 bp (the transcription initiation site of PTCH1a was designated as 0) that contained 19 CpG sites was chosen for bisulfite-sequencing PCR (BSP) and methylation-specific PCR (MSP) detection. The gastric cancer cell line AGS was treated with 5-aza-2’-deoxycytidine (5-Aza-dC; 1 μmol/L) for 3 d. Alterations in PTCH1a TRR methylation in treated AGS cells was measured through BSP clone sequences, and their PTCH1 expression was measured by quantitative RT-PCR. The cell cycle and apoptosis were observed with flow cytometry through propidium iodide (PI) staining or annexin V/PI double staining. The prevalence of PTCH1a TRR methylation was investigated in 170 gastric cancer tissue samples and the adjacent normal tissues by MSP. The correlation of PTCH1a TRR methylation with PTCH1 expression or with patients’ clinical features was analyzed.

RESULTS: Methylation of PTCH1a TRR was observed in AGS cells and a subset of gastric cancer tissues (32%, 55/170), while no methylation amplification products were observed in any normal tissues by MSP. The methylation of PTCH1a TRR was correlated negatively with PTCH1 expression (Spearman’s r = -0.380, P = 0.000). However, methylation of PTCH1a TRR was not related to the gastric cancer patients’ clinical features, such as sex, age of onset, clinical stage, lymph node metastasis or histological grade. The methylation of PTCH1a TRR in AGS cells was almost converted to non-methylation after 5-Aza-dC treatment, which increased PTCH1 expression (5.3 ± 2.5 times; n = 3) and apoptosis rate (3.0 ± 0.26 times; P < 0.05; n = 3).

CONCLUSION: Methylation of PTCH1a TRR is present in a subset of gastric cancers and correlated negatively with PTCH1 expression. This may be an early event in gastric tumorigenesis and a new treatment target.

Keywords: Carcinogenesis, Methylation, Hedgehog signaling pathway, Methylation, PTCH1, Stomach neoplasms