13-顺式维A酸通过CCCTC结合因子促进神经母细胞瘤衰老的作用及机制

13-cis-retinoic acid through CCCTC binding factor promotes the senescence in neuroblastoma and its mechanisms

目的探讨13-顺式维A酸(13-cis RA)对神经母细胞瘤(NB)细胞衰老的调控作用及机制。方法 NB细胞株分为空载体对照组、CCCTC结合因子(CTCF)过表达组、随机干扰组、CTCF干扰组, 筛选稳定过表达、敲低CTCF的亚克隆细胞株;二甲基亚砜(DMSO)、13-cis RA分别处理空载体对照组、CTCF过表达组前后, 采用实时定量PCR、Western blot法检测瘤细胞中CTCF及靶基因的转录水平和蛋白表达变化;噻唑蓝(MTT)比色分析法检测细胞增殖活性;β-Gal原位染色法检测细胞衰老程度的改变。组间比较采用t检验。结果 13-cis RA能抑制NB细胞增殖活性, 促进细胞衰老;CTCF在NB中显著高表达;CTCF过表达组端粒酶逆转录酶(TERT)表达高于对照组(5.32±0.07比1.00±0.02, t=57.17, P<0.05)、Pescadilo 1的表达高于对照组(3.93±0.06比1.00±0.01, t=47.76, P<0.05), 细胞增殖活性增强, 细胞衰老被抑制;在CTCF干扰组, TERT的表达低于对照组(0.35±0.04比1.00±0.01, t=18.26, P<0.05)、PES1表达低于对照组(0.36±0.05比1.00±0.01, t=13.46, P<0.05), 细胞增殖活性降低, 细胞衰老增多;13-cis RA处理后, 瘤细胞中CTCF表达下调, 回补CTCF表达水平能逆转13-cis RA对NB细胞增殖和衰老的调控作用。结论 13-cis RA能下调转录因子CTCF的表达, 抑制衰老相关基因TERT、PES1的表达, 促进NB细胞的衰老。

Objective To explore the regulatory effects and underlying mechanisms of 13-cis retinoic acid(13-cis RA)on senescence of neuroblastoma(NB).Methods NB cells were divided into empty vector group,CCCTC-binding factor(CTCF)over-expression group,scramble shRNA group,and sh-CTCF group,while subclonal cells over-expressing or silencing CTCF were screened.Upon treatment with dimethylsurfoxide(DMSO)or 13-cis RA,the transcript and protein levels of CTCF and target genes were detected by real-time RT-PCR and Western blotting respectively in NB cells stably over-expressing or silencing CTCF.The methyl thiazolyl tetrazolium(MTT)colorimetric andβ-gal staining assays were used to detect the changes in proliferation and senescence of NB cells.Student’s t-test was applied for statistical analysis.Results 13-cis RA inhibited the proliferation and promoted senescence of NB cells.The CTCF was highly expressed in NB cells.In the CTCF overexpression group,the expression of telomerase reverse transcriptase(TERT)and Pescadilo 1(PES1)was higher than that in the control group(TERT:5.32±0.07 vs.1.00±0.02,t=57.17,P<0.05;PES1:3.93±0.06 vs.1.00±0.01,t=47.76,P<0.05).The overexpression of CTCF increased cell proliferation,and reduced senescence of NB cells.In the CTCF interference group,the expression of TERT and PES1 was lower than that of the control group(TERT:0.35±0.04 vs.1.00±0.01,t=18.26,P<0.05;PES1:0.36±0.05 vs.1.00±0.01,t=13.46,P<0.05).Knockdown of CTCF decreased cell proliferation,and enhanced senescence of NB cells.13-cis RA could inhibit the expression of CTCF and rescue the alterations in proliferation and senescence of NB cells induced by stable CTCF over-expression.Conclusion 13-cis RA down-regulates the expression of senescence-related genes TERT and PES1 via CTCF,and promotes the senescence of NB cells.