HA117调节DPF3表达对神经母细胞瘤分化的影响

HA117 inhibits the differentiation via regulation of DPF3 in neuroblastoma

目的 探讨HA117及DPF3在儿童神经母细胞瘤（NB）中的表达及意义,及其对NB分化的影响和可能机制.方法 运用实时荧光定量PCR （qRT-PCR）、免疫组化法检测HA117、DPF3在35例NB、8例节细胞神经母细胞瘤（GNB）和10例神经节细胞瘤（GN）中的表达水平.构建HA117过表达和空载慢病毒,分别转染SH-SY5Y细胞株,构建HA117过表达组和对照组.采用qRT-PCR和Western Blot检测HA117和DPF3的RNA和蛋白质表达水平.采用10 μmol/L维甲酸诱导HA117过表达组和对照组细胞,普通光学显微镜观察细胞形态,免疫荧光法检测神经特异性标志物MAP2蛋白质表达,Western Blot检测HA117过表达组和对照组细胞MAP2、NSE蛋白质的表达情况.结果 HA117的RNA在NB中的相对表达量为30.14±15.96,明显高于GNB/GN的1.03±0.37（P＜0.05）,DPF3的mRNA在NB中的表达量为0,81±0.17,明显低于GNB/GN的3.14±1.09（P＜0.05）,与HA117的表达趋势相反.免疫组化检测发现DPF3在GNB/GN中呈强阳性表达,在分化中的NB中呈弱阳性表达,在未分化/分化差的NB中呈阴性表达.过表达HA117可在mRNA和蛋白水平明显下调SH-SY5Y中DPF3的表达.经10 μmol/L维甲酸诱导7d后,对照组细胞生长明显减慢,细胞长出突起,发生神经元样分化;HA117过表达组SH-SY5Y的MAP2、NSE的表达水平明显低于对照组（P＜0.05）.结论 HA117和DPF3的表达可能与NB的分化相关,HA117可能通过下调DPF3的表达抑制NB的分化.

Objective To investigate the expression and signification of HA117 and DPF3,as well as the effects of them on the differentiation in neuroblastoma.Methods Quantitative reverse transcription-polymerase chain reaction （qRT-PCR） and immunohistochemistry were employed to detect the expression of HA117 and DPF3 mRNA and protein expression in 35 neuroblastomas （NB）,8 ganglioneuroblastomas （GNB） and 10 ganglioneuromas （GN）.HA117 overexpression and control groups were established by lentivirus transduction in SH-SY5Y.qRT-PCR and Western blot were used to detect HA117 and DPF3 mRNA and protein expression level in cells.After SH-SY5Y cells of HA117 overexpression group and control group were induced with 10μmol/L retinoic acid（RA） for 7 days,cell morphology was observed by phase contrast microscop,immunofluorescence was performed to examine MAP2 expression,Western blot was employed for detecting the expression of MAP2 and NSE.Results The RNA levels of HA117 in NB were significantly higher than those in GNB/GN,conversely（30.14 ± 15.96 VS 1.03 ± 0.37,P 〈 0.05）,the mRNA levels of DPF3 in NB were significantly lower than those in GNB/GN （0.81 ± 0.17 VS 3.14 ± 1.09,P 〈 0.05）.The immunohistochemical staining of DPF3 was strongly positive in GNB/GN,weakly positive in differentiating NB and negative in undifferentiated or poorly differentiated NB.The mRNA and protein expression levels were significantly negatively regulated by HA117 overexpression in SH-SY5Y cells.After induced with RA,cells in control group growed slowly and differentiated with synapses.The protein levels of MAP2 and NSE in HA117 overexpression group were significantly lower than those in control group（P〈0.05）.Conclusions The expression of HA117 and DPF3 are related with the differentiation of neuroblastoma,HA117 may inhibits the differentiation of neuroblastoma through regulation of DPF3.