三氧化二砷对维甲酸耐药的神经母细胞瘤细胞SK-N-AS中HoxC9表达的影响

Effect of arsenic trioxide on HOXC9 expression in retinoic acid-resistant neuroblastoma cells SK-N-AS

目的本研究通过检测三氧化二砷(ATO)处理前后维甲酸(RA)耐药的SK-N-AS细胞中HoxC9以及EZH2的表达,初步探寻ATO促进RA耐药的NB细胞分化的具体机制。方法用CCK-8试剂盒测定ATO对NB细胞SK-N-AS增殖抑制率,倒置相差显微镜下观察细胞生长情况,并测量神经突触总长度值。利用质谱技术和非标定量蛋白组学方法检测ATO处理后SK-N-AS细胞蛋白信号丰度变化,并对与NB细胞分化相关蛋白进行差异化分析。Western Blot检测HoxC9、HoxD8及EZH2蛋白的表达情况。结果(1)ATO对SK-N-AS细胞的杀伤作用在4~128μM范围内呈浓度依赖性,ATO对SK-N-AS细胞的半抑制浓度IC50=95.36μM。(2)16μM的ATO处理SK-N-AS细胞24h后,神经突触总长度值升高,与对照组相比差异具有统计学意义(P<0.05)。(3)16μM ATO处理24h后,SK-N-AS细胞内共鉴定到6424个表达蛋白,其中与分化相关的蛋白,上调的包括正常神经元分化标记物MAPT、NEFM等;下调的包括神经母细胞成瘤性标记物PHOX2B。(4)Western Blot结果表明,16μM ATO处理SK-N-AS细胞24h后,HoxC9、HoxD8蛋白的表达水平上调,EZH2蛋白表达水平下调。结论(1)ATO可诱导SK-N-AS细胞的神经突触增多,上调SK-N-AS细胞中神经元正常分化相关的标记物HoxC9及HoxD8的表达,下调神经母细胞成瘤性标记物PHOX2B及EZH2。(2)ATO可能通过下调EZH2重新激活HoxC9的表达。

Objective To obtain specific mechanisms of ATO promoting the differentiation of RA resistant NB cells by detecting the HOXC9 and EZH2 changes in SK-N-AS treated with ATO.Methods Growth inhibition of SK-N-AS cells was assessed using Cell Counting Kit-8(CCK-8).The inverted phase contrast microscope was adopted for observing cell growth and measuring the total length of nerve synapses.To analyze the proteome changes induced by treatment of the SK-N-AS cell with ATO for 24h,the Liquid Chromatography-Mass Spectrometry(LC-MS)was employeed for conducting the label-free proteome quantitation to identify protein intensities information.Western Blot was used to detect the expression of HOXC9,HOXD8 and EZH2.Results(1)ATO inhibited the proliferation of SK-N-AS cells in a dose-dependent manner from 4μM to 128μM and the IC50 of ATO were identified to be 95.36μM.(2)SK-N-AS cells were treated with 16μM ATO for 24h,and the total length of synapses was increased with statistically significant compared with the control group(P<0.05).(3)We identified quantitative information for 6424 proteins where the proteins related to differentiation including MAPT,NEFM and other markers of differentiation with normal neurons and the sympathetic neuroblastic tumorigenic marker PHOX2B were upregulated and downregulated in ATO-treated group.(4)The expression of HOXC9 and HOXD8 were up-regulated and EZH2 was downregulated in ATO-treated group.Conclusions(1)ATO induces the increase of synapses in RA resistant NB cells by upregulating the expressions of HOXC9 and HOXD8.At the same time,the oncogenic marker PHOX2B of neuroblast cells is down-regulated and the markers related to normal differentiation of neurons are up-regulated.(2)ATO may reactivate the expression of HOXC9 by down-regulating EZH2.