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中华乳腺病杂志(电子版)

中华乳腺病杂志(电子版) ›› 2019, Vol. 13 ›› Issue (04) : 225 -233. doi: 10.3877/cma.j.issn.1674-0807.2019.04.007

所属专题： 文献；

论著

乳腺癌HOXA4基因甲基化的检测及其临床意义
李少英1,†(), 麦慧芬1, 黎桂森1, 梁碧婵1, 姜敏1, 甄建新2, 王辉林2, 陈少君3   
  1. 1. 518133　暨南大学附属深圳市宝安区妇幼保健院乳腺科
    2. 518133　暨南大学附属深圳市宝安区妇幼保健院中心实验室
    3. 518000　深圳市妇幼保健院乳腺科
  • 收稿日期:2019-03-19 出版日期:2019-08-01
  • 通信作者: 李少英
  • 基金资助:
    深圳市科技计划资助项目(JCYJ20150403105513703); 广东省科技计划资助项目(20150211); 深圳市三名工程项目出生缺陷防治研究与转化团队项目(201406007)

Detection of aberrant HOXA4 gene methylation in breast cancer and its clinical significance

Shaoying Li1,†(), Huifen Mai1, Guisen Li1, Bichan Liang1, Min Jiang1, Jianxin Zen2, Huilin Wang2, Shaojun Chen3   

  1. 1. Department of Breast Diseases, Bao’an Maternal and Child Health Hospital, Jinan University, Shenzhen 518133
    2. Central Laboratory, Bao’an Maternal and Child Health Hospital, Jinan University, Shenzhen 518133
    3. Department of Breast Diseases, Shenzhen Maternal and Child Health Hospital, Shenzhen 518000, China
  • Received:2019-03-19 Published:2019-08-01
  • Corresponding author: Shaoying Li
  • About author:
    Corresponding author: Li Shaoying, Email:
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引用本文：

李少英, 麦慧芬, 黎桂森, 梁碧婵, 姜敏, 甄建新, 王辉林, 陈少君. 乳腺癌HOXA4基因甲基化的检测及其临床意义[J]. 中华乳腺病杂志(电子版), 2019, 13(04): 225-233.

Shaoying Li, Huifen Mai, Guisen Li, Bichan Liang, Min Jiang, Jianxin Zen, Huilin Wang, Shaojun Chen. Detection of aberrant HOXA4 gene methylation in breast cancer and its clinical significance[J]. Chinese Journal of Breast Disease(Electronic Edition), 2019, 13(04): 225-233.

目的

探讨乳腺癌患者HOXA4基因启动子甲基化的情况及其与临床病理特征的相关性。

方法

运用NEBNext? Ultra? RNA Library Prep Kit for Illumina?进行基因表达芯片测序，运用随机数字表法选择2014年深圳市宝安区妇幼保健院收治的9例乳腺癌患者，分析癌组织与相应癌旁组织异常差异表达的基因。运用Illumina Infinium HD Methylation 450K Assay进行DNA甲基化测序，分析乳腺癌甲基化差异基因。基于肿瘤基因组图谱(The Cancer Genome Atlas，TCGA)数据库信息，分析乳腺癌的差异表达基因和差异甲基化基因。挑选显著高甲基化与低表达的基因，结合生物信息学，确定HOXA4为候选基因。运用随机数字表法收集2014—2017年深圳市宝安区妇幼保健院收治的另外86例乳腺癌患者，采用焦磷酸测序法和RT-PCR，检测乳腺癌组织及其癌旁乳腺组织中HOXA4基因甲基化率和mRNA表达，用Fisher确切概率法分析甲基化率与患者临床病理特征的关系。用Cox比例风险模型进行风险因素的单因素和多因素分析。分别用0、0.5、1、5、10、20 μmol/L的甲基化抑制剂RG108处理乳腺癌MCF-7细胞5 d后，检测HOXA4 mRNA的表达。

结果

基因表达数据芯片分析发现在乳腺癌组织中有1 680个显著上调的基因和1 249个下调基因，整体水平上在不同区域乳腺癌患者甲基化水平较癌旁组织高(P均<0.001)。86例乳腺癌组织中HOXA4基因的甲基化率为94% (81/86)，其中，30例高甲基化，52例低甲基化；而在对应癌旁组织中，HOXA4基因甲基化率为57%(49/86)，其中49例低甲基化，无高甲基化(P<0.001)。有HOXA4甲基化组的癌组织样本中HOXA4 mRNA表达低于无HOXA4甲基化组的癌组织样本(P＝0.003)。HOXA4基因甲基化水平与乳腺癌淋巴结转移、ER表达有关(P＝0.039、0.017)。单因素分析结果显示患者的TNM分期、组织学分级、淋巴结转移及HOXA4甲基化是DFS的危险因素(RR＝4.008，95%CI＝1.296～12.393，P＝0.016；RR＝10.111，95%CI＝2.607～39.217，P＝0.001；RR＝4.588，95%CI＝1.201～17.523，P＝0.026；RR＝1.051，95%CI＝1.007～1.098，P＝0.024)。多因素分析显示组织学分级是乳腺癌患者DFS的独立预后因素(RR＝14.461，95%CI＝2.429～86.100，P＝0.003)。采用不同浓度的RG108来处理MCF-7细胞后，各组HOXA4 mRNA表达比较，差异有统计学意义(χ2＝4.472，P＝0.029)。

结论

HOXA4基因启动子甲基化在乳腺癌的发生、发展中起着重要作用，有潜力作为新的分子生物学指标，用于乳腺癌临床诊断。

关键词: 乳腺肿瘤, 基因，HOXA4, 甲基化
Objective

To investigate the methylation of HOXA4 in breast cancer patients and its correlation with clinicopathological characteristics.

Methods

NEBNext? Ultra? RNA Library Prep Kit for Illumina? was used for gene expression microarray and the screening of abnormally expressed genes in breast cancer tissues and adjacent breast tissues from 9 breast cancer patients (enrolled by a random number table)in Bao’an Maternal and Child Health Hospital in 2014. Illumina Infinium HD Methylation450K Assay was used for DNA methylation microarray and detection of differentially methylated genes in breast cancer. Then the differentially expressed genes and methylated genes in breast cancer were further explored based on The Cancer Genome Atlas (TCGA). The genes with significant hypermethylation and low expression were selected. Combined with bioinformatics, HOXA4 was identified as a candidate gene, with the potential for the detection of early breast cancer. The methylation and mRNA expression of HOXA4 gene in breast cancer tissues and adjacent breast tissues from 86 breast cancer patients (enrolled by a random number table)in Bao’an Maternal and Child Health Hospital from 2014 to 2017 were detected by pyrophosphoric acid sequencing and RT-PCR. And the correlation between HOXA4 mehtylation and the clinicopathological characteristics was also analyzed by the Fisher exact test. Cox proportional hazards model was used for univariate and multivariate analysis of risk factors. The breast cancer MCF-7 cells were treated with 0, 0.5, 1, 5, 10, 20 μmol/L methylation inhibitor RG108 for 5 days, then HOXA4 mRNA expression was detected.

Results

The gene expression microarray showed that 1 680 upregulated genes and 1 249 downregulated genes were determined in breast cancer tissue. The overall methylation levels in different regions of breast cancer tissues were significantly higher than those in adjacent tissues (All P<0.001). In 86 breast cancer patients, the methylation rate of HOXA4 gene was 95% (82/86) in breast cancer tissue(52 samples with low methylation and 30 with hypermethylation), 57% (49/86) in the corresponding adjacent tissues (49 samples with low methylation and none with hypermethylation) (χ2=4.779, P=0.029). The expression of HOXA4 mRNA in HOXA4 methylation group was significantly lower than that in non-methylation group (P=0.031). HOXA4 methylation was correlated with lymph node metastasis(P=0.039) and ER negative (P=0.017). Univariate analysis showed that TNM stage, histological grade, lymph node metastasis and HOXA4 methylation were risk factors for DFS (RR=4.008, 95%CI=1.296-12.393, P=0.016; RR=10.111, 95%CI=2.607-39.217, P=0.001; RR=4.588, 95%CI=1.201-17.523, P=0.026; RR=1.051, 95%CI=1.007-1.098, P=0.024). Multivariate analysis showed that histological grade was an independent prognostic factor for DFS in breast cancer patients (RR=14.461, 95%CI=2.429-86.100, P=0.003). After treatment with different concentrations of RG108, the expression of HOXA4 mRNA in MCF-7 cells was significantly different among six groups (χ2=4.472, P=0.029).

Conclusion

The methylation of HOXA4 plays an important role in the occurrence and development of breast cancer, which may serve as a novel molecular biological marker for clinical diagnosis of breast cancer.

Key words: Breast neoplasms, Gene, HOXA4, Methylation
表1 Cox比例风险回归模型变量赋值表
变量 变量赋值
年龄 <46岁＝0，≥46岁＝1
肿瘤大小 ≤20 mm＝0，>20 mm＝1
TNM分期 Ⅰ、Ⅱ期＝0，Ⅲ期＝1
组织学分级 1、2级＝0，3级＝1
淋巴结转移 无转移＝0，有转移＝1
ER 阴性＝0，阳性＝1
PR 阴性＝0，阳性＝1
HER-2 阴性＝0，阳性＝1
Ki67 <30%＝0，≥30%＝1
TP53 正常＝0，突变＝1
图1 9例乳腺癌组织与癌旁组织差异基因火山图
图2 9例样本的乳腺癌组织及癌旁组织中不同基因区域差异基因甲基化率比较
图3 TCGA数据库中的乳腺癌组织及癌旁组织不同基因区域的差异基因甲基化率比较
图4 9例样本的乳腺癌组织及癌旁组织中不同基因区域HOXA4基因甲基化率比较
图5 差异甲基化基因-差异表达基因-乳腺癌基因相互作用网络
表2 乳腺癌与癌旁乳腺组织中HOXA4基因甲基化情况
癌旁乳腺组织 乳腺癌 合计
有甲基化 无甲基化
有甲基化 49 0 49
无甲基化 32 5 37
合计 81 5 86
表3 乳腺癌HOXA4基因有无甲基化组的mRNA表达情况
组别 低表达 高表达
HOXA4甲基化 30 3
HOXA4无甲基化 0 3
表4 86例乳腺癌患者HOXA4基因甲基化与临床病理特征的关系
临床病理特征 例数 肿瘤组织甲基化例数(%) P值
年龄 ? ? ?
? < 46岁 38 38(100) 0.126
? ≥ 46岁 48 44(92)
淋巴结转移 ? ? ?
? 无 39 35(90) 0.039
? 有 47 47(100)
TNM分期 ? ? ?
? Ⅰ/Ⅱ期 60 56(93) 0.310
? Ⅲ期 26 26(100)
肿瘤大小 ? ? ?
? ≤20 mm 40 37(93) 0.334
? >20 mm 46 45(98)
组织学分级 ? ? ?
? 1～2级 52 48(92) 0.149
? 3级 34 34(100)
ER表达 ? ? ?
? 阴性 54 54(100) 0.017
? 阳性 32 28(88)
PR表达 ? ? ?
? 阴性 39 39(100) 0.123
? 阳性 47 43(91)
HER-2表达 ? ? ?
? 阴性 58 55(95) 1.000
? 阳性 28 27(96)
TP53突变 ? ? ?
? 正常 44 41(93) 0.616
? 突变 42 41(98)
Ki67表达 ? ? ?
? <30% 39 37(95) 1.000
? ≥30% 47 45(96)
表5 86例乳腺癌患者无瘤生存率的单变量分析结果
变量 回归系数 标准误 Wald值 P值 RR 95%置信区间
年龄 －0.598 0.559 1.142 0.285 0.550 0.184～1.647
肿瘤大小 1.213 0.625 3.766 0.052 3.364 0.988～11.451
TNM分期 1.388 0.576 5.812 0.016 4.008 1.296～12.393
组织学分级 2.314 0.692 11.192 0.001 10.111 2.607～39.217
淋巴结转移 1.523 0.684 4.965 0.026 4.588 1.201～17.523
ER －1.120 0.685 2.674 0.102 0.326 0.085～1.249
PR －0.916 0.566 2.624 0.105 0.400 0.132～1.212
HER-2 －0.074 0.596 0.015 0.902 0.929 0.289～2.989
Ki67 0.731 0.590 1.536 0.215 2.078 0.654～6.604
TP53 0.683 0.569 1.437 0.231 1.979 0.648～6.042
HOXA4甲基化 0.050 0.022 5.088 0.024 1.051 1.007～1.098
图6 不同浓度的甲基化抑制剂RG108处理后MCF-7细胞中HOXA4 mRNA的表达
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