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-STAT3）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、2.0 μmol/L时，U266细胞株的总凋亡率分别为0.06%、0.56%、48.96%、61.07%（χ²=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%（χ²=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，进一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 cells

Objective To investigate the effects of arsenic trioxide (AS₂O₃) on SOCS-1 gene methylation and expression of P-STAT3 in multiple myeloma (MM) cells. 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 AS2O3 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 AS₂O₃ 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%(χ²=9.19, P<0.05); and the total cell apoptosisis ratio of CZ-1 was 4.2%,,40.3%,,47.72%,,68.49%(χ²=8.96, P<0.05). Conclusion AS₂O₃could inhibit JAK/STAT signal transduction pathway by inducing SOCS-1 gene demethylation in MM cells which might be related to cell apoptosis.