左旋多巴处理SH-SY5Y细胞可能通过DNA甲基化调节单胺氧化酶B基因的转录

L-DOPA regulates MAO-B transcription through DNA methylation in SHSY5Y cells

目的探究左旋多巴(L-3,4-dihydroxyphenylalanine,L-DOPA)对多巴胺代谢关键基因单胺氧化酶B(monoamine oxidase B,MAO-B)转录的影响及其表观遗传学调控机制。方法以人神经母细胞瘤SH-SY5Y细胞作为研究对象,应用基因组甲基化定量试剂盒检测L-DOPA对基因组甲基化的影响;采用半定量RT-PCR方法探究L-DOPA和DNA甲基转移酶抑制剂5-氮杂-2'-脱氧胞苷(5-aza-2'-deoxycytidine,5-aza-d C)对MAO-B转录的影响;应用甲基化特异性PCR(methylation specific PCR,MSP)技术检测L-DOPA对MAO-B基因启动子区Cp G岛甲基化程度的影响。结果 1L-DOPA(2μmol/L和20μmol/L)和50μmol/L 5-aza-d C处理SH-SY5Y细胞24 h均会使其基因组甲基化降低。2L-DOPA(2μmol/L和20μmol/L)处理SH-SY5Y细胞24 h会使MAO-B mRNA下调,而多巴脱羧酶(DOPA-decarboxylase,DDC)和儿茶酚胺氧位甲基转移酶(catechol-O-methyltransferase,COMT)转录无明显变化。3不同浓度5-aza-d C(0、50、100和150μmol/L)处理细胞24 h,MAO-B mRNA表达明显上调,DDC和COMT转录无明显变化,提示MAO-B可能受到DNA甲基化调控。4L-DOPA处理可使MAO-B基因启动子区Cp G岛甲基化升高,而5-aza-d C可使该区域Cp G岛甲基化降低,说明该区域甲基化受L-DOPA影响而升高,并可能是MAO-B基因转录活性下调的原因。结论 LDOPA可能对MAO-B基因的转录活性有抑制作用,且可能是通过L-DOPA诱导的高Cp G甲基化所导致,从表观遗传学(主要是DNA甲基化)对临床上L-DOPA治疗可能产生的多巴胺代谢紊乱及其不良反应做出了新的解释。

Objective To explore the effect and molecular mechanism of L-3,4-dihydroxyphenylalanine（ L-DOPA） on transcription of monoamine oxidase B（ MAO-B）,which was a key gene in dopamine metabolisms. Methods SH-SY5 Y cells were used as the object to mimic dopaminergic neurons in Parkinson＇s disease. Methylated DNA quantitation kit was used to analyze genomic 5-m C content. The gene transcriptional levels were measured by semi-quantitative RT-PCR. Methylation specific PCR（ MSP） was applied to detect the promotor Cp G methylation status of MAO-B. Results 1 Genomic 5-m C content was decreased in SH-SY5 Y cells treated with L-DOPA（ 2 μmol/L and 20 μmol/L） for 24 h. 2 Monoamine oxidase B（ MAO-B） mRNA level was significantly down-regulation in L-dopa administration,but neither DOPA-decarboxylase（ DDC） nor catechol-O-methyltransferase（ COMT）. 3 A DNA methyltransferase inhibitor,5-aza-2＇-deoxycytidine（ 5-aza-d C）,induced MAO-B mRNA level up-regulation,but COMT and DDC mRNA levels did notaffected. 4 Cp G hyper-methylation of MAO-B was induced by L-DOPA（ 2 μmol / L and 20 μmol / L） for 24 h. And Cp G hypomethylation was induced by 5-aza-d C. Conclusion Transcription of MAO-B was down-regulated by L-DOPA and might be mediated by L-DOPA induced promoter Cp G hyper-methylation. This study might provide a new evidence in the view of epigenetics,especially DNA methylation,on dopamine dysregulation and L-DOPA induced side effects in clinic.