Epigenetic regulation mechanism of abnormally low expression of SHP-1 and its clinical significance in ESCC / 中国肿瘤生物治疗杂志

ABSTRACT

@#[Abstract] Objective: This study aimed at investigating the epigenetic regulation mechanism of abnormally low expression of SHP-1 gene in esophageal squamous cell carcinoma (ESCC). Methods:Atotal of 71 cases of ESCC tissues and corresponding para-cancer tissues(2 cm from the edge of the cancer) resected during surgery at the Department of thoracic surgery of Hebei Province, the Fourth Hospital of Hebei Medical University from 2008 to 2011 were collected for this study. The expression level of SHP-1 mRNA and protein was detected in esophageal cancer cell lines (Eca109, Kyse170, Yes-2) before and after 5-Aza-dC or TSA treatment by RT-qPCR and Western blotting methods respectively. The methylation status of CpG sites in promoter region of SHP-1 was analyzed by bisulfite genome sequencing (BGS) in three esophageal cancer cell lines before and after 5-Aza-dC treatment. The methylation status of SHP-1 was studied by methylation-specific polymerase chain reaction (MSP) method in esophageal cancer cell lines, ESCC tissues and para-cancer tissues. The association between the SHP-1 promoter methylation status and clinic pathological parameters were analyzed in ESCC patients. Dual-luciferase reporter assay systems method was applied to detect the impacts of methylation status of CpG island in SHP-1 promoter region on gene transcription activity. For prognostic analysis of SHP-1 methylation, survival curves were constructed using the Kaplan-Meier method and the log-rank. Results: After treated with 5-Aza-dC, the expression level of SHP-1 mRNA and protein was significantly up-regulated in Eca109, Kyse170 and Yes-2 cells, meanwhile the methylation status of SHP-1 was decreased (P<0.05). The expression level of SHP-1 had no obviously change after treated with trichostatin A(TSA). The methylation frequency of promoter in ESCC tumor tissues was significantly higher than that in corresponding para-cancer tissues (P<0.05). When stratified for clinic pathologic characteristics, methylation frequency of SHP-1 was associated with TNM stage, pathological differentiation, and LN metastasis (P<0.05). The mRNAexpression level of SHP-1 in the ESCC tissues with SHP-1 methylation was significantly decreased compared to the ESCC tissues with unmethylation of SHP-1 (P<0.05). It was associated with methylation of promoter (P<0.05). The activity of fluorescein reporter vector in methylase treatment group was significantly lower than that in untreated group (P<0.05), indicating that SHP-1expression can be silenced by methylation of SHP-1 promoter. The result of Kaplan-Meier shown that SHP-1 promoter methylation was correlated with ESCC patients’poor survival. Conclusion: The transcriptional activity of SHP-1 can be inhibited with hypermethylated SHP-1 promoter region. The hypermethylated SHP-1 promoter induced the silencing of SHP-1. Therefore, SHP-1 gene may serve as one of prognostic methylation biomarkers for ESCC patients.