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

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.

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).

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.