| DNMT1蛋白通过沉默MEG3基因促进视网膜母细胞瘤增殖 |
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| 引用本文： | 高亚莉,罗小玲,孟 婷,朱敏娟,田渼雯,陆晓和. DNMT1蛋白通过沉默MEG3基因促进视网膜母细胞瘤增殖[J]. 南方医科大学学报, 2020, 40(9): 1239-1245. DOI: 10.12122/j.issn.1673-4254.2020.09.03 |
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| 作者姓名： | 高亚莉 罗小玲 孟 婷 朱敏娟 田渼雯 陆晓和 |
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| 摘 要： | 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。
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| 关 键 词： | 视网膜母细胞瘤 DNMT1蛋白 MEG3基因 DNA甲基化 |
DNMT1 protein promotes retinoblastoma proliferation by silencing MEG3 gene |
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| Abstract: | 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) cellline 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 detectthe changes in the expression of DNMT1 protein in the transfected cells, and CCK-8 and EdU assays were used to detect thechanges in cell proliferation. Real-time quantitative PCR (qRT-PCR) was performed to detect MEG3 expression in SO-RB50and HXO-RB44 cells after transfection, and the methylation level of MEG3 gene promoter after interference of DNMT1expression was detected using methylation-specific PCR. Results SO-RB50 and HXO-RB44 cells showed significantly increasedexpression 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 significantlyreduced expression of MEG3 (P<0.05). In SO-RB50 cells, transfection with si-DNMT1 significantly reduced the expression ofDNMT1 protein, suppressed the cell proliferation, and increased MEG3 expression (P<0.05). Interference of DNMT1significantly reduced the methylation level of MEG3 gene promoter. After reversing the regulatory effect of DNMT1 on MEG3gene, DNMT1 protein showed significantly weakened ability to regulate retinoblastoma cell proliferation (P<0.05). ConclusionIn retinoblastoma cells, the up-regulation of DNMT1 protein induces promoter methylation and inactivation of MEG3 geneand eventually leads to abnormal cell proliferation. |
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| Keywords: | retinoblastoma DNMT1 protein MEG3 gene DNA methylation |
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