| Summary: | 碩士 === 國立成功大學 === 藥理學研究所 === 97 === Background and Purpose: The hypermethylated promoter of tumor suppressors leads to gene silence and takes part in tumorigenesis. DNA methyltransferases (DNMTs) mainly include DNMT1, DNMT3A, and DNMT3B, which have been previously reported to be overexpressed in many cancers. However, the transcriptional control of DNMTs in cancer remains mostly unclear. It has been demonstrated that RB and E2F1 repress DNMT1 promoter activity and reduce the DNMT1 protein level. Therefore, this study aims to examine whether the transcriptional regulation of DNMT3A gene is also under the control of RB/E2F1 pathway in cancer using cell and clinical studies.
Results: Using the promoter activity assay, this study found that overexpression of RB inhibited DNMT3A promoter activity. In addition, overexpression of RB decreased mRNA and protein levels of DNMT3A in various cancer cells, whereas si-knockdown of RB increased DNMT3A mRNA and protein levels, suggesting that RB/E2F1 negatively regulated the transcriptional activity of DNMT3A. Using DNA affinity precipitation assay (DAPA), chromatin immunoprecipitation assay and Western blot, the current study showed for the first time that E2F1 bound to DNMT3A promoter region and activated DNMT3A expression. However, RB/E2F1 complex bound on the DNMT3A promoter to act as transcriptional repressors. Moreover, it has been reported that murine double minute (MDM2) facilitates RB degradation. Therefore, the study investigated whether MDM2 plays a role in regulating DNMT3A by degradation of RB in cell model and lung cancer patients. The data indicated that overexpressed MDM2 in cancer cells upregulated mRNA and protein levels of the DNMT3A and decreased the RB protein level. Furthermore, immunoprecipitation showed that RB interacted with MDM2. In addition, overexpression of MDM2 increased the level of RB ubiquitination. Importantly, RB and DNMT3A showed inverse correlation in 106 lung cancer patients (P = 0.016). DNMT3A and MDM2 showed concurrent expression pattern in lung cancer patients (P < 0.001). Patients with normal expression for all tested proteins including DNMT3A, RB and MDM2 showed better post-operative survival than other patients (P = 0.049), indicating that DNMT3A protein expression in relation to RB and MDM2 expression status can be a prognostic factor for lung cancer.
Conclusion: This study provides first cell and clinical evidence that RB/E2F1 pathway transcriptionally represses DNMT3A expression and this regulation can be attenuated by MDM2 expression. In clinical consequence, lung cancer patients with normal expression for DNMT3A, RB and MDM2 proteins have significantly better prognosis compared to other patients. Interestingly, the results of DAPA/MS and Western blot analyses showed that hnRNP A1 protein may cooperate with RB and E2F1 to bind to the DNMT3A promoter and suppress its expression. Further characterization of hnRNP A1 cooperating with RB and E2F1 to repress the DNMT3A transcription is worthy of investigation.
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