The role of ATBF1 nuclear translocation in gastric and intestinal phenotype and chemosensitivity of gastric cancer

背景与目的：MUC5AC属于分泌型粘蛋白家族，在胃小凹细胞中表达。粘蛋白表达在胃癌中发生改变。有报道称MUC5AC在胃癌中的表达与预后不良相关。然而，MUC5AC的转录调控机制尚未得到很好的阐明。AT基序结合因子1 （ATBF1）是一种负向调节甲胎蛋白（AFP）和癌蛋白Myb的同源转录因子。我们之前的研究表明，ATBF1负性调节刷边酶基因氨基肽酶- n的转录，并且ATBF1的缺失是产生afp的胃癌细胞的一个明显特征，其特征是极高的恶性（Oncogene 2001）。ATBF1最近被确定为前列腺癌的候选肿瘤抑制因子（Nat. Genet. 2005）。在本研究中，我们研究了ATBF1对MUC5AC的转录调控。材料与方法：采用免疫组化方法对41例胃癌123个区标本中ATBF1和MUC5AC的表达进行了分析。2. 我们分析了MUC5AC启动子区是否存在AT基序。3. 为了分析ATBF1对MUC5AC的转录调控，我们在MKN45细胞（胃癌细胞）中进行了瞬时转染和双荧光素酶报告试验。4. 为了分析ATBF1在蛋白水平上对MUC5AC的转录调控作用，我们利用共聚焦显微镜检测了ATBF1过表达的MKN45细胞中MUC5AC的蛋白表达。5. 为了评估ATBF1与MUC5AC启动子区域AT基序的结合，我们进行了染色质免疫沉淀（ChIP）。结果:1。只有9%（3/33）的atbf1阳性（核染色）胃癌MUC5AC阳性，76%（68/90）的atbf1阴性（细胞质染色或未染色）胃癌MUC5AC阳性。在MUC5AC阴性的胃癌细胞中，ATBF1核染色倾向于观察到（p< 0.0001）。2. 我们在MUC5AC启动子区检测到AT基序（-1646-1634）。3. 瞬时转染和双荧光素酶报告试验表明，ATBF1抑制MUC5AC启动子的活性（35±1.1%）。4. 70. 转染对照组的MKN45细胞中MUC5AC阳性的比例为7±1.2%，而转染ATBF1的MKN45细胞中MUC5AC阳性的比例为11.3±3.1% （p< 0.0001）。胃癌细胞中过表达的ATBF1抑制MUC5AC内源性蛋白。5. ChIP显示，ATBF1结合MUC5AC启动子的AT基序。结论：ATBF1结合MUC5AC启动子AT基序，负向调控MUC5AC基因在胃癌中的转录。少

Background and aims: MUC5AC belongs to the family of secreted mucins and expresses in foveolar cells of the stomach. Alterations of mucin expression take place in gastric cancer. It was reported that MUC5AC expression in gastric cancer correlates with poor prognosis. However, the transcriptional regulation mechanism of MUC5AC has not been well elucidated. AT motif binding factor 1 (ATBF1) is a homeotic transcription factor which negatively regulates Alpha-fetoprotein (AFP) and oncoprotein Myb. We have showed previously that ATBF1 negatively regulates transcription of brush-border enzyme gene, aminopeptidase-N and that the absence of ATBF1 is a distinct feature of AFP-producing gastric cancer cells, which are characterized by extremely high malignancy (Oncogene 2001). ATBF1 has recently been identified as a candidate of tumor suppressor for prostate cancer (Nat. Genet. 2005). In this study, we investigated the transcriptional regulation of MUC5AC by ATBF1.Materials and Methods: 1. We an … More alyzed ATBF1 and MUC5AC expression by immunohistochemistry in 123 area of 41 gastric cancers specimen. 2. We analyzed whether there is an AT motif in MUC5AC promoter region or not. 3. To analyze the transcriptional regulation of MUC5AC by ATBF1, we performed transient transfection and dual luciferase-reporter assay in MKN45 cells (gastric cancer cells). 4. To analyze the transcriptional regulation of MUC5AC by ATBF1 in protein level, we examined the protein expression of MUC5AC in MKN45 cells which was overexpressed ATBF1 using confocal microscopy. 5. To assess the binding of ATBF1 to the AT motif in MUC5AC promoter region, we performed chromatin immunoprecipitation (ChIP).Results: 1. Only 9% (3/33) of ATBF1-positive(nuclear staining) gastric cancers were MUC5AC positive, and, 76% (68/90) of ATBF1-negative (cytoplasm staining or no staining )gastric cancers were MUC5AC positive. ATBF1 nuclear staining tends to be observed in gastric cancer cells negative for MUC5AC (p<.0001). 2. We detected the AT motif in MUC5AC promoter region (-1646-1634). 3. Transient transfection and dual luciferase-reporter assay demonstrated that ATBF1 suppressed the activity of MUC5AC promoter (35 plus minus 1.1%). 4. 70. 7 plus minus 1.2% of MKN45 cells transfected with control were MUC5AC positice, however only 11.3 plus minus 3.1% of MKN45 cells transfected with ATBF1 were MUC5AC positive (p<.0001). Overexpressed ATBF1 suppressed MUC5AC endogeneous protein in gastric cancer cells. 5. ChIP revealed that ATBF1 binds AT motif of MUC5AC promoter.Conclusion: ATBF1 binds to AT motif of MUC5AC promoter and negatively regulates transcription of the MUC5AC gene in gastric cancer. Less