乳腺癌HOXA4基因甲基化的检测及其临床意义

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

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 TM 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(χ^2=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。

Objective To investigate the methylation of HOXA4 in breast cancer patients and its correlation with clinicopathological characteristics. Methods NEBNext Ultra TM 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)(χ 2=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 (χ^2=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.