DNMT1 和 DNMT3a 的表达受 GLI1 在人胰腺癌细胞中的调控。
Expression of DNMT1 and DNMT3a are regulated by GLI1 in human pancreatic cancer.
机构信息
Department of Gastroenterology, Shanghai 10th People's Hospital, Tongji University, Shanghai, People's Republic of China.
出版信息
PLoS One. 2011;6(11):e27684. doi: 10.1371/journal.pone.0027684. Epub 2011 Nov 14.
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.
背景与目的
作为 Hedgehog 信号通路的必需转录因子，GLI1 在胰腺癌（PC）的发展中发挥重要作用。DNA 甲基转移酶（DNMTs）介导数量的肿瘤相关基因的甲基化。我们的研究旨在探讨 GLI1 与 DNMTs 之间的关系。
方法
通过免疫组织化学（IHC）检测 PC 患者肿瘤组织和相邻正常组织中 GLI1 和 DNMTs 的表达。用环巴胺和 GLI1-siRNA 处理 PANC-1 细胞，用过表达-GLI1 慢病毒载体转染 BxPC-3 细胞。然后通过 qRT-PCR 和 Western blot（WB）分析 GLI1 和 DNMTs 的表达。然后进行染色质免疫沉淀（ChIP）实验，以证明 GLI1 与 DNMT1 结合。最后，当 GLI1 表达改变时，采用嵌套 MSP 评估 APC 和 hMLH1 的甲基化水平。
结果
IHC 结果表明，PC 组织中 GLI1、DNMT1 和 DNMT3a 的表达均高于相邻正常组织（p<0.05）。在 GLI1 表达被环巴胺在 mRNA 和蛋白水平下调（下调 88.1±2.2%，86.4±2.2%）后，DNMT1 和 DNMT3a 的 mRNA 和蛋白水平分别降低了 91.6%±2.2%和 83.8±4.8%、87.4±2.7%和 84.4±1.3%。当进一步通过 siRNA 敲低 GLI1 表达（mRNA 降低 88.6±2.1%，蛋白降低 63.5±4.5%）时，DNMT1 和 DNMT3a 的 mRNA 降低了 80.9±2.3%和 78.6±3.8%，蛋白降低了 64.8±2.8%和 67.5±5.6%。GLI1 基因转染过表达 GLI1（mRNA 增加 655.5±85.9%，蛋白增加 272.3±14.4%），DNMT1 和 DNMT3a 的 mRNA 和蛋白水平分别增加了 293.0±14.8%和 578.3±58.5%、143.5±17.4%和 214.0±18.9%。ChIP 实验表明 GLI1 蛋白与 DNMT1 结合，但不与 DNMT3a 结合。嵌套 MSP 的结果表明，在 PC 中，GLI1 表达影响 APC 的 DNA 甲基化水平，但不影响 hMLH1。
结论
DNMT1 和 DNMT3a 在 PC 中受 GLI1 调控，DNMT1 是其直接靶基因。
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