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

Abstract

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.

DNA methyltransferase expression is associated with cell proliferation in salivary mucoepidermoid carcinoma

“…p16INK4a inhibits cyclin‐dependent kinases (CDK) CDK4 and CDK6, and thereby preventing G1‐to‐S cell‐cycle phase progression; thus, the methylation of p16INK4a is reported cell‐cycle progression 31 . As well as, the inhibition of DNMT1 leads to the activation of p16, RUNX3, SFRP1, and E‐cadherin 29,32–34 . It shows that DNMT could influence the proliferation by leading methylation of suppressor genes that act directly or indirectly in cell cycle.…”

Activation of PAR4 Upregulates p16 through Inhibition of DNMT1 and HDAC2 Expression via MAPK Signals in Esophageal Squamous Cell Carcinoma Cells

“…It is believed that DNMT1 is required for the deactivation of p16 by DNMT1-mediated methylation that may lead to the development of ESCC [ 18 ]. Growing evidence shows that overexpressed HDACs are associated with tumorigenesis in ESCC [ 28 ].…”

“…In the present study, the activation of PAR4 was able to increase p16 expression and decrease DNMT1 and HDAC2 expression in ESCC cells. Therefore, it seems more likely that the effect of PAR4 on DNMT1 and HDAC2 expression results in promoting p16 gene transcription [ 18 , 31 ]. These results suggested that PAR4 was able to upregulate p16 levels through inhibition of DNMT1 and HDAC2 expression in ESCC cells.…”

“…The results demonstrated that activation of PAR4 could decrease the enrichments of DNMT1 and HDAC2 on the p16 promoter in ESCC cells. Suppressing DNMT1 could lead to the activation of the esophageal suppressor gene p16 [ 18 ]. DNMT1 usually forms a corepressor complex with HDACs and represses transcription [ 19 , 31 ].…”

“…The promoter methylation inactivation of p16 gene can increase the risk of ESCC [ 17 ]. Previous studies have demonstrated that DNA methyltransferase 1 (DNMT1) is required for the maintenance of DNA methylation and the deactivation of p16 by DNMT1-mediated methylation that may lead to the development of ESCC [ 18 ]. At promoters, DNA methylation generally precludes transcription directly by blocking the binding of transcriptional activators or indirectly through the recruitment of methyl-binding proteins and corepressor complexes containing histone deacetylases (HDACs), which cooperatively facilitate the formation of heterochromatin [ 19 ].…”