儿童急性淋巴细胞白血病中β-catenin/NF-κB信号通路负调控FPGS基因表达及其在甲氨蝶呤耐药中的机制研究

Methotrexate (MTX) has been widely used as an anticancer agent in the treatment of pediatric acute lymphoblastic leukemia (ALL) and plays an important role in promoting therapy efficacy and preventing extramedullary relapse. Low expression of folylpolyglutamate synthetase (FPGS) is the determinant of MTX resistance in ALL, but the mechanisms of dysregulation of FPGS remain unclear. Bioinformatics analysis showed that there were 2 potential NF-κB RelA binding sites in the human FPGS promoter region. Positive correlation was found between mRNA levels of RelA and FPGS (r=0.351, P<0.0001). Blockade of NF-κB signaling with its inhibitor PDTC led to decreases in FPGS mRNA and protein levels in T-ALL cell line CCRF-CEM. Negative correlation was found between mRNA levels of β-catenin and FPGS (r=－0.332，P=0.038). It was reported that β-catenin can physically complex with NF-κB and result in a reduction of NF-κB DNA binding, transactivation activity,and target gene expression. Based on the above results, we speculated that β-catenin might repress FPGS expression through interacting with NF-κB. Reporter assay ,electrophoretic mobility shift assay (EMSA), and chromatin Immunoprecipitation (ChIP) would be carried out to investigate the relationship between NF-κB and FPGS. Co-Immunoprecipitation (Co-IP) would be used to examine the interation between NF-κB and β-catenin. We would knockdown β-catenin expression by RNA interference. Then, quantitative PCR and western blot would be used to show the changes in FPGS expression. MTT assay,high performance liquid chromatography (HPLC) and flow cytometry (FCM) would be used to demonstrate the roles of β-catenin/ NF-κB/FPGS pathway in MTX resistance. This study may not only contribute to clarifying mechanisms of drug resistance in ALL, but also probably find out novel therapeutic targets.

MTX是儿童ALL治疗的关键药物之一，对联合化疗的疗效和预防髓外复发起着至关重要的作用。FPGS低表达是MTX耐药的重要因素，但低表达的分子机制尚不清楚。我们前期研究发现FPGS启动子区存在NF-κB RelA的结合位点，二者表达水平呈显著正相关，抑制NF-κB活性后FPGS表达下调；FPGS与β-catenin表达水平呈显著负相关。研究表明β-catenin可与RelA相互作用，抑制RelA的转录激活及其靶基因表达。本研究拟采用报告基因分析、ChIP、EMSA，证明RelA对FPGS的转录调控。采用免疫共沉淀、RNA干扰、报告基因分析、EMSA，证明β-catenin通过与RelA相互作用，抑制FPGS表达。采用RNA干扰、流式细胞术、高效液相色谱、MTT，阐明β-catenin/NF-κB/FPGS通路对MTX耐药的影响。有助于揭示儿童ALL MTX耐药的分子机制，提供新的治疗靶点。

背景：儿童急性淋巴细胞白血病（ALL）是儿童期最常见的恶性肿瘤。目前儿童ALL耐药机制尚不清楚，一些新的融合基因还未发现，从而限制其治愈率的进一步提高。甲氨蝶呤（MTX）是儿童ALL治疗的关键药物之一，有些患儿使用MTX后会发生耐药或不良毒副作用。FPGS低表达是MTX耐药的重要因素，但低表达的分子机制尚不清楚。.研究内容： 1. 在T-ALL细胞系Jurkat中采用RNAi、免疫共沉淀、染色质免疫共沉淀、Western、报告基因等技术研究β-catenin/NF-κB RelA通路对FPGS转录水平是否具有调控作用。2. 分析了MTX转运体相关基因的基因多态性与MTX毒副作用、血药浓度及患儿预后的关系。3. 在儿童ALL中采用PCR方法对前期发现的新的融合基因CREBBP-ZNF384及EP300-ZNF384进行了筛查。.结果：1. 染色质免疫共沉淀及报告基因实验结果显示NF-κB RelA可以转录激活FPGS的表达。免疫共沉淀结果显示β-catenin与NF-κB RelA之间存在相互作用。β-catenin敲降后FPGS蛋白水平表达升高，提示β-catenin可能通过与NF-κB RelA相互作用，从而负调控FPGS，导致其低表达。2. 相关性分析结果显示SLCO1B1 rs10841753与大剂量MTX给药后48小时的MTX血药浓度显著相关（P=0.017）。多因素Cox回归分析结果显示SLCO1B1 rs4149056和SLC19A1 rs2838958是儿童ALL无事件生存的独立预后因素。3. EP300-ZNF384及CREBBP-ZNF384在本中心儿童ALL中的发生频率分别为2.0%及1.8%。.结论：1. 本研究发现β-catenin可能通过与NF-κB RelA相互作用，对FPGS表达进行负调控，从全新的角度探讨了FPGS在T-ALL中低表达的调控机制，具有较重要的科学意义。2. 研究提示儿童ALL中基因多态性能够影响MTX的血药浓度及药物反应，可能为MTX药物反应提供预测分子标志物，具有一定的应用前景。3. 本研究验证了ZNF384融合基因在中国儿童ALL中存在一定的比例，具有较重要的科学意义及应用前景。