[DNMT1 protein promotes retinoblastoma proliferation by silencing MEG3 gene].

OBJECTIVE

To investigate whether DNMT1 protein induces retinoblastoma proliferation by silencing MEG3 gene.

METHODS

Two retinoblastoma cell lines (HXO-RB44 and SO-RB50) and a normal human retinal pigment epithelial (RPE) cell line were transfected with the plasmid pcDNA-DNMT1 or si-DNMT1 for up-regulating or interference of DNMT1 expression, and with pcDNA-MEG3 or si-MEG3 for up-regulating or interference of MEG3 expression. Western blotting was used to detect the changes in the expression of DNMT1 protein in the transfected cells, and CCK-8 and EdU assays were used to detect the changes in cell proliferation. Real-time quantitative PCR (qRT-PCR) was performed to detect MEG3 expression in SO-RB50 and HXO-RB44 cells after transfection, and the methylation level of MEG3 gene promoter after interference of DNMT1 expression was detected using methylation-specific PCR.

RESULTS

SO-RB50 and HXO-RB44 cells showed significantly increased expression of DNMT1 protein as compared with normal RPE cells ( < 0.05). In HXO-RB44 cells, transfection with pcDNADNMT1 resulted in significantly increased expression of DNMT1 protein, enhanced cell proliferation ability, and significantly reduced expression of MEG3 ( < 0.05). In SO-RB50 cells, transfection with si-DNMT1 significantly reduced the expression of DNMT1 protein, suppressed the cell proliferation, and increased MEG3 expression ( < 0.05). Interference of DNMT1 significantly reduced the methylation level of MEG3 gene promoter. After reversing the regulatory effect of DNMT1 on MEG3 gene, DNMT1 protein showed significantly weakened ability to regulate retinoblastoma cell proliferation ( < 0.05).

CONCLUSIONS

In retinoblastoma cells, the up-regulation of DNMT1 protein induces promoter methylation and inactivation of MEG3 gene and eventually leads to abnormal cell proliferation.