Hypomethylation of ETS transcription factor binding sites and upregulation of PARP1 expression in endometrial cancer

Abstract

Although PARP1 promoter methylation is involved in the regulation of PARP1 expression in human keratinocyte lines and lymphoblastoid cell lines, its roles in human endometrial cancer are unknown. DNA from forty normal endometrium (NE) and fifty endometrial adenocarcinoma (EAC) tissues were analyzed by bisulfite sequencing using primers focusing on the core promoter region of PARP1. Expression levels of PARP1 were assessed by immunohistochemistry and real-time PCR. Associations between patient clinicopathological characteristics and PARP1 protein levels were assessed by Fisher's exact test. Here, PARP1 mRNA and protein were overexpressed in EAC tissues (P < 0.05). CpG sites within the ETS motif in the PARP1 promoter exhibited significant hypomethylation in EAC tissues, and there was a significant negative correlation between PARP1 mRNA levels and the number of methylated sites in both NE and EAC tissues (R (2) = 0.262, P < 0.001). Notably, PARP1 protein expression was associated with FIGO stage (P = 0.026), histological grade (P = 0.002) , and body mass index (P = 0.04). Our findings imply that PARP1 overexpression may participate in endometrial cancer progression, and abnormal hypomethylation of CpG sites within the ETS motif in the core promoter region may be responsible for PARP1 overexpression in EAC tissues.

Figure 1

Overexpression of PARP1 protein and mRNA in EAC tissues. (a) Quantification of relative PARP1 mRNA levels. (b(i)) Sections were subjected to immunostaining for PARP1. Arrow denotes positive staining for PARP1 in the nuclei. (b(ii)) Summary of scoring the percentage of positive cells from the measurements shown in (b(i)). Bar graphs show mean ± SE. *P < 0.05 versus NE. Magnification is 400x. NE, normal endometrium; EAC, endometrial adenocarcinoma.

Figure 2

Hypomethylation of the ETS transcription factor binding sites in the promoter of PARP1 in EAC tissues. (a) Location of PARP1 core promoter CpG sites. Genomic coordinates are shown, along with the primer-amplified fragments, GC percentage, location of individual CpG dinucleotides (dashes), CpG island (green bar), and the PARP1 RefSeq gene (exon 1 shown as a blue box and intron shown as an arrowed line). The arrow indicates the transcriptional direction. (b) Changes in methylation patterns in the core promoter region of PARP1. The circles correspond to CpG sites denoted by the black dashes in (a). Closed circles, methylation; open circles, unmethylation. Ten individual clones were sequenced for each sample. (c) Summary of the methylated sites in a CpG within the ETS motif in NE and EAC tissues. Bar graphs show mean ± SE. *P < 0.05 versus NE. (d) Correlation between the relative PARP1 mRNA levels and the number of methylated sites in a CpG within the ETS motif for each sample. Open circles, NE; closed circles, EAC; NE, normal endometrium; EAC, endometrial adenocarcinoma.