title: Expression of DNMT1 and DNMT3a are regulated by GLI1 in human pancreatic cancer.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  DNMT1 and DNMT3a are regulated by GLI1 in PC, and DNMT1 is its direct target gene.
authors: Department of Gastroenterology, Shanghai 10th People's Hospital, Tongji University, Shanghai, People's Republic of China.
authors: ShanShan
authors: S
authors: He
authors: Feng
authors: F
authors: Wang
authors: LiJuan
authors: L
authors: Yang
authors: ChuanYong
authors: C
authors: Guo
authors: Rong
authors: R
authors: Wan
authors: AiWu
authors: A
authors: Ke
authors: Ling
authors: L
authors: Xu
authors: GuoYong
authors: G
authors: Hu
authors: XuanFu
authors: X
authors: Xu
authors: Jie
authors: J
authors: Shen
authors: XingPeng
authors: X
authors: Wang
mesh_terms: D011125:Adenomatous Polyposis Coli / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics; D000090205:DNA Methyltransferase 3A; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics; D006801:Humans; D015513:Oncogene Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D015534:Trans-Activators / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D014704:Veratrum Alkaloids / Q000494:pharmacology; D000071676:Zinc Finger Protein GLI1
chemical_list: C000717967:DNMT3A protein, human; D015513:Oncogene Proteins; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014704:Veratrum Alkaloids; D000071676:Zinc Finger Protein GLI1; D000076123:DNA (Cytosine-5-)-Methyltransferase 1; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; C000619380:DNMT1 protein, human; C000541:cyclopamine