5-氮-2'-脱氧胞苷对肺癌A549细胞凋亡及范可尼贫血互补基团F表达的影响

doi:10.3969/j.issn.1673-8640.2015.05.022

检验医学 ›› 2015, Vol. 30 ›› Issue (5): 507-511.DOI: 10.3969/j.issn.1673-8640.2015.05.022

5-氮-2'-脱氧胞苷对肺癌A549细胞凋亡及范可尼贫血互补基团F表达的影响

余宗涛¹, 张吉才¹, 高琼², 高波¹, 胡春卉¹

1. 湖北医药学院附属十堰市太和医院检验科,湖北十堰 442000
2. 湖北医药学院附属十堰市太和医院呼吸内科,湖北十堰 442000

收稿日期:2014-06-11 出版日期:2015-05-30 发布日期:2015-06-17
作者简介:null
作者简介:余宗涛,男,1971年生,硕士,副主任技师,主要从事肿瘤分子机制研究。

通讯作者:高琼,联系电话:0719-8801750。
基金资助:
湖北省十堰市科技局资助项目(ZD2012015);湖北医药学院优秀中青年科技创新项目(2011CXG02)

Influence of 5-Aza-CdR on the apoptosis of lung cancer A549 cell and the expression of FANCF gene

YU Zongtao¹, ZHANG Jicai¹, GAO Qiong², GAO Bo¹, HU Chunhui¹

1. Department of Clinical Laboratory, Taihe Hospital, Hubei University of Medicine, Hubei Shiyan 442000, China
2. Department of Respiratory Medicine, Taihe Hospital, Hubei University of Medicine, Hubei Shiyan 442000, China

Received:2014-06-11 Online:2015-05-30 Published:2015-06-17

摘要/Abstract

摘要： 目的探讨5-氮-2'-脱氧胞苷(5-Aza-CdR)对人肺癌A549细胞凋亡及抑癌基因范可尼贫血互补基团F(FANCF)基因表达的影响。方法以浓度为0.5、5、50 μmol/L的5-Aza-CdR处理人肺癌细胞株A549,常规培养采用四唑盐(MTT)比色法观察细胞的生长活性,甲基化特异性聚合酶链反应(PCR)检测FANCF基因甲基化状态;以荧光定量PCR检测FANCF mRNA的表达,并用流式细胞仪检测细胞凋亡率。结果 5-Aza-CdR能明显抑制肿瘤细胞的生长,细胞增殖抑制率(CPIR)随5-Aza-CdR浓度和作用时间的不同而变化,呈剂量依赖性(P<0.005)和时间依赖性(P<0.001);药物处理后FANCF mRNA表达明显升高,细胞凋亡率与5-Aza-CdR剂量呈正相关(r=0.998, P<0.05)。结论 5-Aza-CdR能使FANCF基因去甲基化,促进细胞凋亡,增强抑癌功能,但同时有增加顺铂耐药的风险。

关键词: 范可尼贫血互补基团F基因, 甲基化, 5-氮-2'-脱氧胞苷, A549细胞, 凋亡

Abstract: Objective To investigate the influence of 5-Aza-2'-deoxycytidine(5-Aza-CdR) on the apoptosis of lung cancer A549 cell and the expression of Fanconi anemia complementation group F(FANCF) gene. Methods A549 cells were treated with 5-Aza-CdR(0.5, 5 and 50 μmol/L, respectively). The growth of A549 cells was observed by 3-(4,5-dimethylthiazol)-3,5-diphenyltetrazolium bromide(MTT) assay. The methylation status of FANCF gene was observed by methylation specific polymerase chain reaction(PCR). The expression of FANCF mRNA was observed by fluorescence quantitation PCR. The apoptosis rate of A549 cells was analyzed by flow cytometry. Results A549 cells treated with 5-Aza-CdR displayed a slow growth. The rate of cell proliferation inhibiting (CPIR) for A549 cells changed with the concentration and treatment time of 5-Aza-CdR (P<0.005, P<0.001). FANCF mRNA expression increased after treatmert. The apoptosis rates after treatment had a positive correlation with 5-Aza-CdR dose (r=0.998, P<0.05). Conclusions 5-Aza-CdR can induce the apoptosis of A549 cells by inducing demethylation and thereby enchancing FANCF gene, enhancing tumor suppressor function, but it can increase the risk of resistance to cisplatin.

Key words: Fanconi anemia complementation group F gene, Methylation, 5-Aza-2'-deoxycytidine, A549 cell, Apoptosis

中图分类号:

R446.62

余宗涛, 张吉才, 高琼, 高波, 胡春卉. 5-氮-2'-脱氧胞苷对肺癌A549细胞凋亡及范可尼贫血互补基团F表达的影响[J]. 检验医学, 2015, 30(5): 507-511.

YU Zongtao, ZHANG Jicai, GAO Qiong, GAO Bo, HU Chunhui. Influence of 5-Aza-CdR on the apoptosis of lung cancer A549 cell and the expression of FANCF gene[J]. Laboratory Medicine, 2015, 30(5): 507-511.

图/表 5

参考文献 9

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基因名称	上游引物(5'-3')	下游引物(5'-3')	扩增片段长度 (bp)
FANCF-M^[3]	TTTTTGCGTTTGGAGAATCGGGTTTTC	ATACACCGCAAACCGCCGACGAACAAAACG	153
FANCF-U	TTTTTGTGTTTGTTGGAGAATTGGGTTTTT	ATACACCACAAACCACCAACAAACAAAACA	153
FANCF mRNA	TTCGGAAGTCTTTGCTGCCT	AGTAATAACACAGCATTGCC	413
GAPDH mRNA^[4]	GAAGGTGAAGGTCGGAGTC	GAAGATGGTGATGGGATTTC	226

基因名称	上游引物(5'-3')	下游引物(5'-3')	扩增片段长度 (bp)
FANCF-M^[3]	TTTTTGCGTTTGGAGAATCGGGTTTTC	ATACACCGCAAACCGCCGACGAACAAAACG	153
FANCF-U	TTTTTGTGTTTGTTGGAGAATTGGGTTTTT	ATACACCACAAACCACCAACAAACAAAACA	153
FANCF mRNA	TTCGGAAGTCTTTGCTGCCT	AGTAATAACACAGCATTGCC	413
GAPDH mRNA^[4]	GAAGGTGAAGGTCGGAGTC	GAAGATGGTGATGGGATTTC	226

5-Aza-CdR (μmol/L)	CPIR
5-Aza-CdR (μmol/L)	培养24 h	培养48 h	培养72 h
0.5	5.02±2.18^*#△▲	17.97±3.63^*#	27.24±1.79^*#
5	18.06±1.30^△▲	33.22±4.12	39.27±4.02
50	23.11±4.80^△▲	44.33±2.17	62.54±2.04

5-Aza-CdR (μmol/L)	CPIR
5-Aza-CdR (μmol/L)	培养24 h	培养48 h	培养72 h
0.5	5.02±2.18^*#△▲	17.97±3.63^*#	27.24±1.79^*#
5	18.06±1.30^△▲	33.22±4.12	39.27±4.02
50	23.11±4.80^△▲	44.33±2.17	62.54±2.04

5-Aza-CdR (μmol/L)	FANCF mRNA相对定量值
5-Aza-CdR (μmol/L)	培养24 h	培养48 h	培养72 h
0	0.028 6±0.010 0	0.026 0±0.018 9	0.070 3±0.057 4
0.5	0.159 5±0.100 5^*	0.218 4±0.120 9^*	0.247 9±0.117 3^*
5	0.230 7±0.070 1^*	0.321 1±0.141 4^*	0.416 7±0.129 1^*
50	0.461 3±0.140 2^*#	0.652 4±0.198 2^*#	1.207 1±0.226 9^*#

5-氮-2'-脱氧胞苷对肺癌A549细胞凋亡及范可尼贫血互补基团F表达的影响

Influence of 5-Aza-CdR on the apoptosis of lung cancer A549 cell and the expression of FANCF gene

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