研究背景及目的：抑癌基因的啟動子被過度甲基化時，會導致該基因不表達進而使腫瘤生成；此甲基化現象主要是由DNA甲基轉移酵素（DNA 5’-cytosine-methyltransferase，DNMTs）所調控，DNMTs主要有DNMT1、DNMT3A以及DNMT3B，本實驗室及許多文獻指出，在很多癌症中發現DNMTs有過度表達的情形，然而造成此現象的詳細機制仍不清楚；文獻亦顯示RB/E2F1能負調控DNMT1啟動子的活性，進而降低DNMT1蛋白的表現，但對DNMT3A是否具有類似的調控機制尚未清楚。因此本篇研究目的為以細胞及臨床模式探討DNMT3A其轉錄調控異常之機制。
研究方法及結果：首先在細胞層次上，觀察抑癌蛋白 RB 是否對 DNMTs 扮演負轉錄調控因子的角色。由報導基因冷光酵素活性分析（luciferase assay）發現在膀胱癌細胞株5637細胞中大量表現RB後，RB的確會抑制 DNMT3A啟動子的活性及mRNA表現量，並且以西方墨點法（Western blot）發現，RB的過度表達也會降低肺癌細胞株A549及H1299、骨癌細胞株Saos2與膀胱癌細胞株5637的DNMT3A蛋白質表現量；以siRNA降低RB的表現量後，DNMT3A的mRNA與蛋白質表現量上升，顯示RB的確能負轉錄調控DNMT3A的基因表現。
在文獻中已知DNMT1啟動子上含有E2F1蛋白的結合位置，並且知道E2F1能正向調控DNMT1啟動子活性，但是DNMT3A啟動子是否也含有E2F1蛋白的結合區域及是否受E2F1正向調控還尚未知道，所以首先在H1299細胞中，分別大量表現E2F1及用siRNA降低E2F1的表現，發現DNMT3A能受到E2F1的正調控；此外，以DNA親和性沉澱分析（DNA affinity purification assay，DAPA）與染色質免疫沈澱（chromatin immunoprecipitation）等實驗證實當RB與E2F1蛋白同時結合在DNMT3A啟動子上時，會負調控DNMT3A的基因表現。
此外，文獻顯示MDM2會促進RB蛋白與20S proteasome結合或增加RB被ubiquitination之程度，造成RB蛋白降解，所以本研究也觀察MDM2降解RB蛋白的現象是否也在DNMT3A基因調控中扮演重要的角色，進而在H1299及5637細胞中以西方墨點法及免疫沉澱法（immunoprecipitation）實驗證實，過度表現MDM2蛋白會增加DNMT3A蛋白質表現量，也提高了RB蛋白被ubiqutination程度，並且降低了RB蛋白的表現量，使得DNMT3A的轉錄調控異常。
在臨床研究上，以免疫組織染色法（immunohistochemistry）分析106位非小細胞肺癌病人組織中DNMT3A與RB蛋白之相關性，發現DNMT3A蛋白過度表現且RB蛋白低表現之負相關性 （P值為0.016）；MDM2蛋白過度表現且RB蛋白低表現之負相關性（P值為0.042）。此外，病人存活率分析資料指出：DNMT3A、RB與MDM2蛋白皆正常表現的病人，與其他病人相較，有較好的預後（P值為0.049），顯示DNMT3A、RB與MDM2蛋白的表現程度可做為肺癌病人預後的指標。
結論: 由以上細胞及臨床模式實驗結果顯示，E2F1能正轉錄調控DNMT3A啟動子，增加其蛋白質表現量，而RB/E2F1複合體則會負轉錄調控DNMT3A的啟動子，進而降低DNMT3A的mRNA及蛋白質表現，但此RB/E2F1負調控DNMT3A的作用可被MDM2蛋白所拮抗。

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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