三氧化二砷诱导骨髓瘤细胞SOCS-1基因去甲基化及其对P—STAT3蛋白表达的影响

目的探讨三氧化二砷（AS2O3）对多发性骨髓瘤（MM）细胞内细胞因子信号转导抑制因子-1（SOCS-1）基因甲基化状态的影响及其对磷酸化的信号转导与转录激活因子-3（P．STA33）表达的影响。方法采用甲基特异性PCR法检测AS2O3作用前后MM细胞株U266和CZ-1细胞内SOCS-1基因的甲基化状态，应用蛋白免疫印迹法检测AS2O3处理前后细胞内P—STAT3蛋白的表达变化，并采用流式细胞技术检测AS2O3作用前后MM细胞增殖和凋亡的变化。结果MM细胞株内SOCS-1基因存在程度不同的甲基化状态，与对照组相比，AS2O3作用后MM细胞内SOCS-1基因甲基化程度明显减弱或消失，P—STAT3蛋白的表达也明显减弱，同时细胞生长受抑，凋亡比率升高。AS2O3浓度分别为0、0．5、1．0、2．0μmol／L时，U266细胞株的总凋亡率分别为0．06％、0．56％、48．96％、61．07％（x2=9．19，P〈0．05）；而CZ-1细胞株的总凋亡率分别为4．20％、40．30％、47．72％、68．49％（X2=8．96，P〈0．05）。结论AS2O3可能通过诱导MM细胞内SOCS-1基因去甲基化作用，进-步抑制细胞增殖信号Janus激酶（JAK）-STAT通路的活化，从而诱导MM细胞的凋亡。

Effects of arsenic trioxide on intracelluar SOCS-1 gene methylation and P-STAT3 expression in multiple myeloma ceils

Objective To investigate the effects of arsenic trioxide （ AS2 03 ） on SOCS-1 gene methyla- tion and expression of P-STAT3 in multiple myeloma （MM） ceils. Methods MM cell lines U266 and CZ-1 were used as in vitro models. Methylation status of SOCS-1 gene was detected by the methylation specific PCR （MSP）while P-STAT3 protein expression was determined by Western blotting assay before and after AS2O3 treatment. Meanwhile growth inhibition and apoptosis of MM cells were determined by flow cytometry. Results Hypermethylation of SOCS-1 gene was observed in each MM cell line compared with wide type. After exposure to AS2O3 , it was shown that SOCS-1 gene was demethylated obviously, meanwhile the expression level of P-STAT3 protein and cell proliferation was inhibited significantly in each cell line. The apoptosis rate was increased. When U266 and CZ-1 were treated with AS203 of 0,0.5,1.0,：2.0 μmol/L respectively, the total cell apoptosisis ratio of U266 was 0.06% ,0.56% ,48.96% ,61.07% （ X2 = 9.19, P 〈 0.05 ） ; and the total cell apoptosisis ratio of CZ＇-I was 4.2%, ,40.3%, ,47.72%, ,68.49% （ X2 = 8.96, P 〈 0.05）. Conclusion AS203 could inhibit JAK/STAT signal transduction pathway by inducing SOCS-1 gene demethylation in MM cells which might be related to cell apoptosis.