DNMT1 mediates chemosensitivity by reducing methylation of miRNA-20a promoter in glioma cells

Although methyltransferase has been recognized as a major element that governs the epigenetic regulation of the genome during temozolomide (TMZ) chemotherapy in glioblastoma multiforme (GBM) patients, its regulatory effect on glioblastoma chemoresistance has not been well defined. This study investigated whether DNA methyltransferase (DNMT) expression was associated with TMZ sensitivity in glioma cells and elucidated the underlying mechanism. DNMT expression was analyzed by western blotting. miR-20a promoter methylation was evaluated by methylation-specific PCR. Cell viability and apoptosis were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and TdT-mediated dUTP-biotin nick end labeling assays, respectively. The results showed that compared with parental U251 cells, DNMT1 expression was downregulated, miR-20a promoter methylation was attenuated and miR-20a levels were elevated in TMZ-resistant U251 cells. Methyltransferase inhibition by 5-aza-2′-deoxycytidine treatment reduced TMZ sensitivity in U251 cells. In U251/TM cells, DNMT1 expression was negatively correlated with miR-20a expression and positively correlated with TMZ sensitivity and leucine-rich repeats and immunoglobulin-like domains 1 expression; these effects were reversed by changes in miR-20a expression. DNMT1 overexpression induced an increase in U251/TM cell apoptosis that was inhibited by the miR-20a mimic, whereas DNMT1 silencing attenuated U251/TM cell apoptosis in a manner that was abrogated by miR-20a inhibitor treatment. Tumor growth of the U251/TM xenograft was inhibited by pcDNA-DNMT1 pretreatment and boosted by DNMT1-small hairpin RNA pretreatment. In summary, DNMT1 mediated chemosensitivity by reducing methylation of the microRNA-20a promoter in glioma cells.

[1]  Claudia Petritsch,et al.  miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells , 2008 .

[2]  A. Jeltsch,et al.  DNMT1-associated DNA methylation changes in cancer , 2015, Cell cycle.

[3]  Brett L Carlson,et al.  Induction of MGMT expression is associated with temozolomide resistance in glioblastoma xenografts. , 2009, Neuro-oncology.

[4]  M. R. Matarazzo,et al.  De novo DNMTs and DNA methylation: novel insights into disease pathogenesis and therapy from epigenomics. , 2014, Current pharmaceutical design.

[5]  L. Hood,et al.  DNA methyltransferase-mediated transcriptional silencing in malignant glioma: a combined whole-genome microarray and promoter array analysis , 2009, Oncogene.

[6]  S. Golden,et al.  How a cyanobacterium tells time. , 2008, Current opinion in microbiology.

[7]  T. Boon,et al.  Transient Down-regulation of DNMT1 Methyltransferase Leads to Activation and Stable Hypomethylation of MAGE-A1 in Melanoma Cells* , 2006, Journal of Biological Chemistry.

[8]  P. Jones,et al.  DNA methylation errors and cancer. , 1996, Cancer research.

[9]  Stijn van Dongen,et al.  miRBase: tools for microRNA genomics , 2007, Nucleic Acids Res..

[10]  Chang-Zheng Chen,et al.  MicroRNAs as oncogenes and tumor suppressors. , 2005, The New England journal of medicine.

[11]  Y. You,et al.  MicroRNA-125b-2 confers human glioblastoma stem cells resistance to temozolomide through the mitochondrial pathway of apoptosis. , 2011, International journal of oncology.

[12]  Xin Li,et al.  miR-20a targets BNIP2 and contributes chemotherapeutic resistance in colorectal adenocarcinoma SW480 and SW620 cell lines. , 2011, Acta biochimica et biophysica Sinica.

[13]  M. Wolter,et al.  MicroRNA-mediated down-regulation of NKG2D ligands contributes to glioma immune escape , 2014, Oncotarget.

[14]  Hui Zhou,et al.  Inhibition of miR-17 and miR-20a by oridonin triggers apoptosis and reverses chemoresistance by derepressing BIM-S. , 2014, Cancer research.

[15]  Dajiang Xie,et al.  LRIG1 dictates the chemo-sensitivity of temozolomide (TMZ) in U251 glioblastoma cells via down-regulation of EGFR/topoisomerase-2/Bcl-2. , 2013, Biochemical and biophysical research communications.

[16]  Nathaniel H. Boyd,et al.  Inhibition of HDAC1 and DNMT1 Modulate RGS10 Expression and Decrease Ovarian Cancer Chemoresistance , 2014, PloS one.

[17]  S. Ropero,et al.  A microRNA DNA methylation signature for human cancer metastasis , 2008, Proceedings of the National Academy of Sciences.

[18]  Rahima Patel,et al.  Silencing DNA methyltransferase (DNMT) enhances glioma chemosensitivity. , 2008, Oligonucleotides.

[19]  R. Siebert,et al.  Epigenetic silencing of the tumor suppressor microRNA Hsa-miR-124a regulates CDK6 expression and confers a poor prognosis in acute lymphoblastic leukemia. , 2009, Cancer research.

[20]  Baohui Liu,et al.  LRIG1 Enhances Chemosensitivity by Modulating BCL-2 Expression and Receptor Tyrosine Kinase Signaling in Glioma Cells , 2014, Yonsei medical journal.

[21]  M. Esteller,et al.  DNA methylation-associated silencing of tumor-suppressor microRNAs in cancer , 2011, Oncogene.

[22]  Xuejun Yang,et al.  Mechanism of thalidomide to enhance cytotoxicity of temozolomide in U251-MG glioma cells in vitro. , 2009, Chinese medical journal.

[23]  T. Tuschl,et al.  Mechanisms of gene silencing by double-stranded RNA , 2004, Nature.

[24]  Steven J. Greco,et al.  Temozolomide resistance in glioblastoma occurs by miRNA-9-targeted PTCH1, independent of sonic hedgehog level , 2015, Oncotarget.

[25]  Bin Wang,et al.  Oncogenic miR-20a and miR-106a enhance the invasiveness of human glioma stem cells by directly targeting TIMP-2 , 2014, Oncogene.

[26]  Donald C. Chang,et al.  The MicroRNA (miRNA): Overview of the RNA Genes that Modulate Gene Function , 2007, Molecular biotechnology.