Methylation of TFPI-2 is an Early Event of Esophageal Carcinogenesis

Abstract

Aims: To explore the epigenetic changes and the function of TFPI-2 in esophageal cancer. Materials & methods: Nine esophageal cancer cell lines, nine normal esophageal mucosa, 60 esophageal dysplasia and 106 advanced esophageal cancer samples were included in this study. TFPI-2 methylation was examined by methylation-specific PCR. TFPI-2 expression was evaluated by immunohistochemistry in tissue samples. The effect of TFPI-2 on proliferation, apoptosis, invasion and migration was analyzed by colony formation assay, western blot assay, transwell assay and flow cytometric analysis. Results: TFPI-2 expression was regulated by promoter region hypermethylation in human esophageal cancer cell lines, and TFPI-2 expression is inversely correlated with methylation in primary cancer. Methylation was found in 28.2, 33.3 and 33.3% of grade 1, 2 and 3 esophageal dysplasia, and 67% of primary esophageal cancer, but no methylation was found in normal mucosa. Methylation is significantly related to tumor differentiation. Inhibition of invasion, migration, colony formation and proliferation, and induction of apoptosis occurred with the restoration of TFPI-2 expression in the KYSE70 cell line. Conclusion:TFPI-2 is frequently methylated in esophageal cancer with a progression tendency. TFPI-2 is a potential tumor suppressor in esophageal cancer.

KEYWORDS::

• carcinogenesis
• DNA methylation
• dysplasia
• esophageal cancer
• TFPI-2

Financial & competing interests disclosure

This work was supported by grants from the National Basic Research Program (973 Program No. 2012CB934002, 2010CB912802 and 2009CB521801), National HighTechnology R&D Program of China (863 Program No. SS2012AA020821, SS2012AA02A203 and SS2012AA02A209), National Key Scientific instrument Special Programme of China (Grant No. 2011YQ03013405) and National Science Foundation of China (Grant No. 81121004, 81071953 and 81161120432). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Additional information

Funding

This work was supported by grants from the National Basic Research Program (973 Program No. 2012CB934002, 2010CB912802 and 2009CB521801), National HighTechnology R&D Program of China (863 Program No. SS2012AA020821, SS2012AA02A203 and SS2012AA02A209), National Key Scientific instrument Special Programme of China (Grant No. 2011YQ03013405) and National Science Foundation of China (Grant No. 81121004, 81071953 and 81161120432). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.