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

Abstract

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.

Figure 1

Characterization of miR-20a expression in glioma cells. The glioma cell lines U251 and U251/TM were used to determine (a) DNMT profiles using western blotting, (b) miR-20a promoter methylation using methylation-specific PCR (MS-PCR) and (c) miR-20a expression using quantitative RT-PCR. The glioma cell lines U87 and U87/TM were used to determine the expression of (d) DNMTs, (e) miR-20a promoter methylation and (f) miR-20a. Data were represented as the mean±s.d. *P<0.05 compared with the parental U87 or U251 cells.

Figure 2

5-aza-dC treatment reduced the TMZ sensitivity of U251 cell. U251 cells were incubated with 5 μmol l^-1 5-aza-dC, a methyltransferase inhibitor prior to treatment with or without TMZ. (a) Quantification of miR-20a normalized to U6 RNA. (b) Quantification of LRIG1 mRNA normalized to NADPH and representation of LRIG1 protein expression. (c) Cell viability was determined using the MTT assay. Data were represented as the mean±s.d. *P<0.05 compared with the control.

Figure 3

Negative correlation between DNMT1 and miR-20a expression in U251/TM cells. U251/TM cells were transfected with control pcDNA-DNMT1 or pcDNA and then lysed for detection of (a) miR-20a and (b) LRIG1 mRNA and protein expression. (c) Cells were co-treated with pcDNA-DNMT1 and sh-LRIG1, followed by a series of concentrations of TMZ. Then, cell viability was examined. U251/TM cells were transfected with DNMT1-shRNA or control-shRNA and then lysed for analysis of (d) miR-20a and (e) LRIG1 mRNA and protein expression levels. (f) Post-shRNA transfection, the cells were treated with a series of concentrations of TMZ and cell viability was examined. Data were represented as the mean±s.d. *P<0.05 compared with the control plasmid or negative control.

Figure 4

Abrogating effect of miR-20a on DNMT1-stimulated LRIG1 expression in U251/TM cells. (a) U251/TM cells were co-transfected with pcDNA-DNMT1 and the miR-20a mimic or its negative control (NC). Forty-eight hours post transfection, the cells were lysed and the LRIG1 mRNA and protein expression levels were determined. (b) U251/TM cells were co-transfected with DNMT1-shRNA and miR-20a inhibitor or its negative control (NC). Forty-eight hours post transfection, the cells were lysed and LRIG1 mRNA and protein expression levels were determined. (c) Cells were transfected with the miR-20a mimic or (d) miR-20a inhibitor, and 48 h post transfection the cells were transfected with the luciferase reporter gene plasmid that included the miR20-a binding site in the LRIG1 3′-UTR sequence. Luciferase activity and the mRNA and protein expression of LRIG1 were determined. Data were represented as the mean±s.d. *P<0.05 compared with the control plasmid, Pre-NC; ^#P<0.05 compared with the negative control.

Figure 5

Abrogating effect of miR-20a on DNMT1-stimulated cell apoptosis in U251/TM cells. After transfection of U251/TM cells with pcDNA-DNMT1 and/or the miR-20a mimic, the cells were incubated with TMZ (5 μmol l^-1) and lysed for analysis of (a) G1 phase and (b) S phase using the BrdU assay and (c) cell apoptosis using the TUNEL assay. U251/TM cells were transfected with DNMT1-shRNA and/or the miR-20a inhibitor prior to TMZ (5 μmol l^-1) treatment. (d) The cells in the G1 phase, (e) S phase and (f) levels of cell apoptosis were determined as described above. Data were represented as the mean±s.d. ^$P<0.05 compared with no TMZ treatment, *P<0.05 compared with the control plasmid+TMZ, ^#P<0.05 compared with the negative control+TMZ.

Figure 6

Effect of the DNMT1-miR-20a axis on tumor TMZ sensitivity in U251/TM xenografts. (a) Twenty-four hours after co-transfection with pcDNA-DNMT1 and/or the miR-20a mimic or pcDNA, U251/TM cells were transplanted into mice and tumor growth was measured from day 1 until day 21 post xenograft. During this time, the tumors were injected with TMZ (50 mg kg^-1 per day) for 5 days when their volumes reached 0.5 cm. (b) Twenty-four hours after co-transfection with DNMT1-shRNA and/or the miR-20a inhibitor or it negative control, U251/TM cells were transplanted into mice. Tumor growth was measured from day 1 until day 21 post xenograft. During this time, the tumors were injected with TMZ (50 mg kg^-1 per day) for 5 days when their volume reached 0.5 cm. Data were represented as the mean±s.d. *P<0.05 compared with the control, ^#P<0.05 compared with plasmid+TMZ or negative control+TMZ; ^$P<0.05 compared with pc3.1DNA-DNMT1 or sh-DNMT1.