Silencing DNA methyltransferase 1 leads to the activation of the esophageal suppressor gene p16 in vitro and in vivo.

Abstract

Previous studies have demonstrated that DNA methyltransferase 1 (DNMT1) is required for the maintenance of DNA methylation and epigenetic changes that may lead to the development of esophageal squamous cell carcinoma (ESCC). In order to investigate whether the silencing of DNMT1 protects tumor suppressor genes, including p16, and is able to be used as a potential therapy for human ESCC, short hairpin RNA targeting DNMT1 (shRNA-DNMT1) was synthesized and transfected into the human ESCC lines KYSE150 and KYSE410, which were then injected into the backs of nude mice prior to harvesting. Results from the reverse transcription-quantitative polymerase chain reaction (PCR) and western blotting demonstrated that p16 mRNA expression was increased in the shRNA-DNMT1-transfected ESCC cell lines in vitro and in vivo. Consistent with the immunohistochemistry results, p16 was expressed in tumor tissue from nude mice that had been transplanted with the modified human ESCC lines. It was also observed that p16 methylation was inhibited following transfection with shRNA-DNMT1 as detected using methylation-specific PCR analysis. The results of the present study suggest that silencing DNMT1 serves a protective role through the demethylation and subsequent activation of p16 in vitro and in vivo.