Expression of DNMT1 and DNMT3a Are Regulated by GLI1 in Human Pancreatic Cancer

Abstract

Background and AimsGLI1, 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.MethodsExpressions 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.ResultsIHC 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.ConclusionDNMT1 and DNMT3a are regulated by GLI1 in PC, and DNMT1 is its direct target gene.

High expression of DNA methyltransferases in primary human medulloblastoma

“…Our results indicate that SHH pathway activation is associated with elevated DNMT1 expression, which could be a consequence of upregulation of DNMT1 by SHH in SHH-associated medulloblastomas. Similar observations were made in pancreatic cancer and in myelodysplastic syndrome (MDS) [16,40]. DNMT1 and DNMT3A could be regulated by Gli1, an effector of the SHH signaling pathway.…”

“…DNMT1 and DNMT3A could be regulated by Gli1, an effector of the SHH signaling pathway. The SHH pathway was inhibited in pancreatic cancer and MDS cell lines, resulting in decreased DNMT1 expression, whereas induction of the SHH pathway resulted in higher DNMT1 expression [16,40]. In contrast, DNMT1 inhibition resulted in increased expression of SFRP1 in MB cell lines [20].…”

“…Overexpression of DNMTs was detected at the mRNA or protein level. Increased DNMT1 expression was described in gastric cancers, along with DNMT3A in breast and pancreatic cancer, or along with DNMT3B in glioma [8,16,30,38,39]. Although there is emerging evidence that epigenetic events play a remarkable role in the development of adult cancers, our knowledge about their role in pediatric brain tumors is still very limited.…”

DNMT3a rs1550117 Polymorphism Association with Increased Risk of Helicobacter pylori Infection

“…In addition, the over-expression of DNMT3a mRNA and protein were detected in various cancers including GC, these results revealed that increased DNMT3a expressiom may play a role in tumorgenesis of GC (Oh et al, 2007;Ding et al, 2008;Rahman et al, 2010;He et al, 2011;Yang et al, 2011). However, no differences of DNMT3a expression was detected between different genotype carriers in GC patients in our study.…”

“…Also, allelic loss of DNMT3a has been frequently observed in many common solid cancers, including GC, lymphomas and lung cancers (Kim et al, 2013). Several studies have found that DNMT3a is over-expressed in human cancers, suggesting that it may be involved in tumorgenesis and tumor progression (Ding et al, 2008;He et al, 2011;Yang et al, 2011). Recently, DNMT3a SNPs rs1550117 and rs13420827 were also found to be significantly associated with GC (Fan et al, 2010;Wu et al, 2012;Yang et al, 2012).…”