Promoter hypermethylation of ARID1A gene is responsible for its low mRNA expression in many invasive breast cancers

Abstract

ARID1A (AT-rich interactive domain 1A) has recently been identified as a tumor suppressor gene. Its mRNA expression is significantly low in many breast cancers; this is often associated with more aggressive phenotypes. However, the underlying molecular mechanism for its low expression has not been fully understood. This study was undertaken to evaluate the contribution of gene copy number variation, mutations, promoter methylation and histone modification to ARID1A's low expression. 38 pairs of breast invasive ductal carcinomas and their normal breast tissue counterparts from the same patients were randomly selected for gene expression and copy number variation detection. Promoter methylation and histone modification levels were evaluated by MeDIP-qPCR and ChIP-qPCR, respectively. PCR product Sanger sequencing was carried out to detect the exon mutation rate. Twenty-two out of 38 invasive ductal carcinomas in the study (57.9%) revealed ARID1A mRNA low expression by realtime RT-PCR. The relative promoter methylation level was, significantly higher in ARID1A mRNA low expression group compared with its high expression group (p<0.001). In the low expression group, nineteen out of 22 invasive ductal carcinomas (86.4%) exhibited ARID1A promoter hypermthylation. In addition, the promoter hypermethylation was accompanied with repressive histone modification (H3K27Me3). Although five out of 38 invasive ductal carcinomas (13.2%) exhibited loss of ARID1A gene copy number by realtime PCR and nine exon novel mutations are seen from eight out of 33 invasive ductal carcinomas (24.2%), there was no statistically significant difference in both ARID1A mRNA low and high expression groups (p=0.25,and p=0.68, respectively). We demonstrate that promoter hypermethylation was the main culprit for ARID1A mRNA low expression in invasive ductal carcinomas. The influence of mutation and copy number variation on the expression were statistically insignificant at mRNA level, and were, therefore, not considered the main causes for ARID1A mRNA low expression in invasive breast cancer.

Figure 1. ARID1A promoter methylation.

CpG site and island around transcription start site (TSS) were predicted by Methyl Primer Express. Two primer sets P1 and P2 were designed by Primer Primier 5 to amplify the TSS upstream CpG island.

Figure 2. ARID1A gene promoter methylation levels between RNA high (n = 11) and low (n = 22) expression groups.

Relative methylation levels were analyzed with MeDIP-qPCR. Promoter methylation levels (blue bar) were significantly higher in low expression group (green bar) compared with high expression group (green bar) (p<0.001).

Figure 3. ARID1A gene promoter histone modification levels between RNA high (n = 5) and low (n = 5) expression groups.

The histone modifications of H3K27Ac, H3K27Me3 and H3K4Me3 were analyzed with ChIP-qPCR. Only repressive marker H3K27Me3 was significantly higher (p<0.001) in low expression group compared with high expression group.

Figure 4. Copy number variations between ARID1A low and high expression groups.

Samples were divided into low (n = 22) and high (n = 11) expression groups based on ARID1A mRNA relative expression (green). The difference of relative copy number variation (blue) was not significant between two groups (p = 0.274).

Figure 5. ARID1A mutation sites in genome and corresponding exon.

(A). Three mutation sites indentified with Sanger sequencing in three cancer (CA) and corresponding normal (PY) tissues. (B). All the mutation sites indentified from our research were indicated in genome. Sample information, normal (green round spot) or cancer (red round spot) tissue, RNA low (green square) or high (red square) expression were also indicated. (C). Mutation sites in exon. Protein synonymous (green star) and nonsynonymous (red star) mutations were indicated. Two of the mutation sits located in ARID domain.

Figure 6. Different contributions to ARID1A mRNA abnormal expression.

ARID1A relative mRNA, methylation, mutations and copy number variations were presented from outer to inner in doughnut chart. Percentage was indicated with length of circular arc.