DNMT1蛋白通过沉默MEG3基因促进视网膜母细胞瘤增殖

DNMT1 protein promotes retinoblastoma proliferation by silencing MEG3 gene

目的探讨DNMT1蛋白是否通过沉默MEG3基因诱导视网膜母细胞瘤增殖。方法通过转染pcDNA-DNMT1或si-DNMT1上调或干扰DNMT1的表达水平;通过转染pcDNA-MEG3或si-MEG3上调或干扰MEG3的表达水平;用Western blot检测视网膜母细胞瘤细胞系中DNMT1蛋白表达量;用CCK-8法及EdU法检测细胞的增殖能力;用实时荧光定量PCR技术检测转染后的视网膜母细胞瘤细胞中MEG3表达量的变化;干扰DNMT1表达后,用甲基化特异性PCR检测MEG3基因启动子DNA甲基化水平的变化。结果视网膜母细胞瘤SO-RB50及HXO-RB44细胞中DNMT1蛋白表达量显著升高(P<0.05)。转染了pcDNA-DNMT1的HXO-RB44细胞中DNMT1蛋白表达增加,细胞增殖能力增加,MEG3表达量降低;转染了siRNADNMT1的SO-RB50细胞DNMT1蛋白表达减少,细胞增殖能力降低,MEG3表达量增加(P<0.05)。干扰DNMT1蛋白表达后,MEG3基因启动子DNA甲基化水平降低(P<0.05)。逆转DNMT1蛋白对MEG3基因的调控后,DNMT1蛋白调控RB细胞增殖的能力减弱(P<0.05)。结论在视网膜母细胞瘤细胞中,DNMT1蛋白表达的上调,诱导了MEG3基因启动子DNA甲基化失活,最终导致细胞异常增殖。

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(P<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(P<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(P<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(P<0.05).Conclusion In retinoblastoma cells,the up-regulation of DNMT1 protein induces promoter methylation and inactivation of MEG3 gene and eventually leads to abnormal cell proliferation.