Hypermethylation of MEG3 promoter correlates with inactivation of MEG3 and poor prognosis in patients with retinoblastoma

Abstract

Background: In our previous study, we revealed that MEG3 was a tumor suppressor gene in retinoblastoma and inhibited proliferation of retinoblastoma cells by regulating the activity of the Wnt/β-catenin pathway. Here, we further explored the mechanism of MEG3 inactivation in retinoblastoma.

Methods: MSP and qRT-PCR were performed to detect the methylation status of MEG3 promoter and levels of MEG3 expression, respective. To further explore relationship between MEG3 expression and epigenetic modifications, 5-Aza-CdR was used to interfere with DNA methylation. In addition, we evaluated proliferation, apoptosis and the expression of β-catenin via CCK-8, flow cytometric analysis and western blot analysis, respective.

Results: Hypermethylation of MEG3 promoter was observed more frequently in retinoblastoma tissues and was highly associated with low MEG3 expression and poor survival of retinoblastoma patients. We also provided evidence demonstrating that hypermethylation of MEG3 promoter depressed MEG3 expression, promoted proliferation, inhibited apoptosis and increased β-catenin expression of retinoblastoma cells in vitro.

Conclusions: Our present study indicates that promoter silencing by hypermethylation may account for the loss of MEG3 expression and predict poor prognosis.

Keywords: DNA methylation; Epigenetic; Long non-coding RNA; MEG3; Prognosis; Retinoblastoma.

Fig. 1

The level and clinical significance of MEG3 methylation in retinoblastoma. a The methylated (160 bp) and unmethylated (120 bp) PCR products were designated as M or U, respectively. Retinoblastoma tissues C1–C5 and corresponding non-tumor tissues N1–N5 was used as examples. b The frequency of methylation of MEG3 promoter in retinoblastoma tissues and corresponding non-tumor tissues was shown. c Correlation of promoter methylation and MEG3 expression in retinoblastoma. d and e Kaplan–Meier plots for patient’s survival according to the methylation status of MEG3 promoter. The log-rank test was used to calculate the P value. **P < 0.01

Fig. 2

The effect of 5-Aza-CdR on MEG3 methylation and MEG3 expression in Weri-Rb1 and Y79 cells. a Hypermethylation of MEG3 promoter was observed in Weri-Rb1 and Y79 cells. 5-Aza-CdR decreased the methylation level of MEG3 promoter. b 5-Aza-CdR down-regulated the expression level of MEG3. *P < 0.05, **P < 0.01

Fig. 3

The effect of 5-Aza-CdR on cell proliferation and Wnt/β-catenin pathway in Weri-Rb1 and Y79 cells. a 5-Aza-CdR inhibited the cell proliferation. b 5-Aza-CdR depressed the activity of the Wnt/β-catenin pathway. *P < 0.05

Fig. 4

The effect of 5-aza-CdR on cell apoptosis in Weri-Rb1 and Y79 cells. a and b 5-Aza-CdR increased the number of early apoptotic cells of Weri-Rb1 and Y79 cells. *P < 0.05, **P < 0.01

Fig. 5

MEG3 re-expression played an important role in the antitumor effect of 5-Aza-CdR in Weri-Rb1 and Y79 cells. a Si-MEG3 was just offset the MEG3 re-expression effect of 5-Aza-CdR. b Si-MEG3 could block the effect of 5-Aza-CdR on β-catenin expression. c Si-MEG3 could reverse the effect of 5-Aza-CdR on cell proliferation. *P < 0.05

Fig. 6

MEG3 re-expression contributed to inhibitory effect of 5-aza-CdR on apoptosis in Weri-Rb1 and Y79 cells. a and b Si-MEG3 could depress the effect of 5-Aza-CdR on cell apoptosis. *P < 0.05