Expression of DNMT1 and DNMT3a are regulated by GLI1 in human pancreatic cancer.
Expression of DNMT1 and DNMT3a are regulated by GLI1 in human pancreatic cancer.
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人胰腺癌中 DNMT1 和 DNMT3a 的表达受 GLI1 调节。
DOI：
10.1371/journal.pone.0027684
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Wang X
中科院分区：
文献类型：
--
作者：
He S;Wang F;Yang L;Guo C;Wan R;Ke A;Xu L;Hu G;Xu X;Shen J;Wang X
背景与目的GLI 1作为Hedgehog信号通路中不可或缺的转录因子，在胰腺癌的发生发展中起重要作用。DNA甲基转移酶（DNMTs）介导大量肿瘤相关基因的甲基化。本研究旨在探讨GLI 1与DNMT的关系。方法采用免疫组织化学方法检测PC患者肿瘤及癌旁正常组织中GLI 1和DNMTs的表达。PANC-1细胞用环巴胺和GLI 1-siRNA处理，而BxPC-3细胞用过表达GLI 1慢病毒载体转染。然后通过qRT-PCR和western blot（WB）分析GLI 1和DNMTs的表达。然后，我们采取染色质免疫沉淀（ChIP）证明GLI 1结合DNMT 1。最后，采用巢式MSP评估GLI 1表达改变时APC和hMLH 1的甲基化水平。结果GLI 1、DNMT 1和DNMT 3a在PC组织中的表达均高于癌旁正常组织（p<0.05）。环巴胺抑制GLI 1 mRNA和蛋白表达（分别下调88.1± 2.2%和86.4± 2.2%）后，DNMT 1和DNMT 3a mRNA和蛋白表达分别下降91.6%±2.2%和83.8± 4.8%，87.4±2.7%和84.4± 1.3%。当siRNA进一步下调GLI 1表达（mRNA下降88.6± 2.1%，蛋白下降63.5±4.5%）时，DNMT 1和DNMT 3a mRNA分别下降80.9±2.3%和78.6±3.8%，蛋白下降64.8±2.8%和67.5± 5.6%。GLI 1基因转染后GLI 1的过表达（mRNA增加655.5± 85.9%，蛋白增加272.3± 14.4%），DNMT 1和DNMT 3a mRNA和蛋白表达分别增加293.0±14.8%和578.3± 58.5%，143.5±17.4%和214.0± 18.9%。ChIP检测显示GLI 1蛋白与DNMT 1结合，但不与DNMT 3a结合。巢式MSP结果显示，GLI 1表达影响APC的DNA甲基化水平，但不影响hMLH 1。结论DNMT 1和DNMT 3a在PC中受GLI 1的调控，DNMT 1是其直接靶基因。
Background and Aims GLI1, as an indispensable transcriptional factor of Hedgehog signaling pathway, plays an important role in the development of pancreatic cancer (PC). DNA methyltransferases (DNMTs) mediate the methylation of quantity of tumor-related genes. Our study aimed to explore the relationship between GLI1 and DNMTs. Methods Expressions of GLI1 and DNMTs were detected in tumor and adjacent normal tissues of PC patients by immunohistochemistry (IHC). PANC-1 cells were treated by cyclopamine and GLI1-siRNA, while BxPC-3 cells were transfected with overexpression-GLI1 lentiviral vector. Then GLI1 and DNMTs expression were analyzed by qRT-PCR and western blot (WB). Then we took chromatin immunoprecipitation (ChIP) to demonstrate GLI1 bind to DNMT1. Finally, nested MSP was taken to valuate the methylation levels of APC and hMLH1, when GLI1 expression altered. Results IHC result suggested the expressions of GLI1, DNMT1 and DNMT3a in PC tissues were all higher than those in adjacent normal tissues (p<0.05). After GLI1 expression repressed by cyclopamine in mRNA and protein level (down-regulation 88.1±2.2%, 86.4±2.2%, respectively), DNMT1 and DNMT3a mRNA and protein level decreased by 91.6%±2.2% and 83.8±4.8%, 87.4±2.7% and 84.4±1.3%, respectively. When further knocked down the expression of GLI1 by siRNA (mRNA decreased by 88.6±2.1%, protein decreased by 63.5±4.5%), DNMT1 and DNMT3a mRNA decreased by 80.9±2.3% and 78.6±3.8% and protein decreased by 64.8±2.8% and 67.5±5.6%, respectively. Over-expression of GLI1 by GLI1 gene transfection (mRNA increased by 655.5±85.9%, and protein increased by 272.3±14.4%.), DNMT1 and DNMT3a mRNA and protein increased by 293.0±14.8% and 578.3±58.5%, 143.5±17.4% and 214.0±18.9%, respectively. ChIP assays showed GLI1 protein bound to DNMT1 but not to DNMT3a. Results of nested MSP demonstrated GLI1 expression affected the DNA methylation level of APC but not hMLH1 in PC. Conclusion DNMT1 and DNMT3a are regulated by GLI1 in PC, and DNMT1 is its direct target gene.
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影响因子：
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作者：
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影响因子：
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