Abstract 3132: Suppressor microRNA-145 is epigenetically regulated by promoter hypermethylation in esophageal squamous cell carcinoma
Authors: harada kazuto , Baba Yoshifumi , Kosumi Keisuke , Tokunaga Ryuma , Izumi Daisuke , Ouchi Mayuko , Nakamura Kennichi , Kiyozumi Yuki , Kurashige Junji , Hiyoshi Yukiharu , Iwagami Shiro , Miyamoato Yuji , Sakamoto Yasuo , Yoshida Naoya , Watanabe Masayuki , Baba Hideo 
Published: August 2015

Abstract
Objectives: DNA methylation change is common epigenetic change in cancer. The promoter CpG island hypermethylation is one of the most common mechanisms by which tumor suppressor miRNAs are inactivated during tumorigenesis. However, microRNA (miRNA) regulations by epigenetic alteration, especially CpG island hypermethylation, have been not yet substantially understood in esophageal squamous cell carcinoma (ESCC). Therefore, we performed the screening of miRNAs which should be up-regulated after 5-aza-20-deoxycytidine (5-Aza) treatment in ESCC cell lines, then from up-regulated miRNAs, we focused on a suppressor miR-145-5p. Since miRNA-145-5p attracts an increasing attention as a tumor suppressor miRNA in ESCC, we evaluated miRNA-145 promoter methylation level and miRNA-145-5p expression level in ESCC
Methods: The screening of miRNAs was analyzed using the Human miFinder 384HC miScript miRNA PCR Array. DNA methylation level was evaluated by using bisulfite-pyrosequencing assay. miRNA expression level was evaluated by using TaqMan quantitative real-time reverse transcription-polymerase chain reaction. miRNA-145-5p expression and its promoter methylation levels were quantified in 15 ESCC and matched 15 normal adjacent esophageal mucosa frozen tissues and in 105 formalin-fixed paraffin-embedded ESCC tissues between September 2009 and June 2012 at Kumamoto University Hospital (Kumamoto, Japan).
Results Twenty miRNAs including miR-145-5p were up-regulated more than 3 fold in both TE-1 and KYSE30 after 5-Aza treatment by using miRNA PCR array analysis. After 5-Aza treatment, miRNA-145 promoter methylation level decreased and the expression level of miRNA-145-5p was significantly enhanced in 8 of 10 ESCC cell lines. The miRNA-145-5p expression levels in the cancer tissues (mean ± SD; 6.37 ± 1.47) was significantly lower than in matched normal adjacent esophageal epithelial mucosa (235.6 ± 61.9) (p = 0.0024). Moreover, the miRNA-145 promoter in the cancer tissues (41.4 ± 3.05) was significantly hyper methylated than in matched normal adjacent epithelial mucosa (mean; 57.1 ± 3.36) (p = 0.0042 by the paired t test). In 105 ESCC samples, the “high miRNA-145-5p expression level group” and “low miRNA-145-5p expression level group” experienced similar overall survival (Kaplan-Meier analysis; log rank p = 0.58) and disease-free survival (Kaplan-Meier analysis; log rank p = 0.80)
Conclusions: Our results suggest that hypermethylation of the miRNA-145 promoter region might regulate miRNA-145 expression in ESCC. This is the first report on the epigenetic regulation of miRNA-145 in ESCC. However, further work is needed to assess our results and to make them more definite.
Citation Format: kazuto harada, Yoshifumi Baba, Keisuke Kosumi, Ryuma Tokunaga, Daisuke Izumi, Mayuko Ouchi, Kennichi Nakamura, Yuki Kiyozumi, Junji Kurashige, Yukiharu Hiyoshi, Shiro Iwagami, Yuji Miyamoato, Yasuo Sakamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. Suppressor microRNA-145 is epigenetically regulated by promoter hypermethylation in esophageal squamous cell carcinoma. [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 3132. doi:10.1158/1538-7445.AM2015-3132