Abstract 4768: Paired box 2 (PAX2) is involved in carcinogenesis and regulated by DNA methylation in endometrial cancer

Abstract

Abstract Objective: PAX2 is recognized as a significant factor in the development of urogenital system. It is expressed in multiple tumors and is essential in tumor cell survival. This study aimed to investigate the effect, expression, methylation of PAX2 in endometrial cancer, and the possible regulative mechanism of DNA methylation related PAX2 expression. Methods: PAX2 expression in endometrial cancer, hyperplasia and normal tissues was detected by immunohistochemistry, and its expression in cell lines was measured by real-time PCR, western blot and immunocytochemistry. The role of PAX2 on cell viability and mobility was measured by CCK-8, invasion and migration assays. Bisulfite sequencing and MassARRAY were used to quantify the DNA methylation level of PAX2 promoter in endometrial cells and tissues. Deletion analysis of PAX2 promoter was performed by luciferase reporter gene to analyze its promoter activity. Transcription factors (TFs) combined with PAX2 promoter were forecasted by TF search database and the regulatory function of these TFs were measured by detecting the expression of PAX2 mRNA and protein following the knock down of these TFs. Results: PAX2 was over-expressed in endometrial cancer tissues compared to hyperplasia and normal endometrium. Compare to normal endometrial epithelial cells, endometrial cancer cell lines exhibited moderate to high PAX2 expression. Knocking down PAX2 by siRNA reduced cell viability, invasion and migration, while PAX2 over-expression exhibited opposite role. The methylation level of PAX2 promoter was higher than normal counterparts in both cancer tissues and cells, which was positively related to PAX2 expression. After 5-Aza-CdR treatment, the PAX2 mRNA and protein was down-regulated along with the methylation level of PAX2 promoter. Deletion analysis confirmed that a repressive transcriptional regulatory region of PAX2 promoter coincided with the hypermethylated region found in MassARRAY analysis. The binding sites of myeloid zinc finger 1 (MZF1) and the early growth responsive gene 1(EGR-1) were found in the repressive transcriptional regulatory region of PAX2 promoter. Knocking down MZF1 or EGR-1 upregulated both the mRNA and protein level of PAX2 after 5-Aza-CdR treatment, which indicated that MZF1 and EGR-1 may act as repressive transcription factors in the DNA methylation related PAX2 transcription. Conclusion: PAX2 is involved in carcinogenesis of endometrial cancer by stimulating cell growth and promoting cell motility. Overexpression of PAX2 in endometrial cancer may be regulated by promoter hypermethylation via repressive transcription factors such as MZF1 or EGR-1. Citation Format: Nan Jia, Jieyu Wang, Qing Li, Xiang Tao, Keqin Hua, Yinhua Yu, Jan P. Baak, Kwong Kwok Wong, Weiwei Feng. Paired box 2 (PAX2) is involved in carcinogenesis and regulated by DNA methylation in endometrial cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4768. doi:10.1158/1538-7445.AM2015-4768