Abstract 5195: Claudin-1, a novel target of miR-375 in non-small cell lung cancer
Authors: Yoda Satoshi , Soejima Kenzo , Yasuda Hiroyuki , Sato Takashi , Arai Daisuke , Ohgino Keiko , Ishioka Kota , Tani Tetsuo , Oashi Ayano , Kuroda Aoi , Nishino Makoto , Miyawaki Masayoshi , Hamamoto Junko , Naoki Katsuhiko , Betsuyaku Tomoko 
Published: May 2015

MicroRNAs, the small and noncoding RNAs, regulate the translation of specific protein-coding genes. Accumulated evidence strongly suggests that microRNAs play important and complex roles in human cancers, including lung cancer. We previously reported that miR-375 expression was low in squamous cell carcinoma (SCC) and high in adenocarcinoma (AC) of lung cancer. The target gene of miR-375 in non-small cell lung cancer (NSCLC) has not been elucidated. The purpose of this study was to identify a target of miR-375 and clarify the function of miR-375 in NSCLC. Candidate genes of miR-375 targets were determined using the prediction databases and also previous findings about the different gene expression between SCC and AC. We focused on claudin-1 (CLDN1), which has four putative target sites of miR-375 in its 3′-untranslated region (UTR). CLDN1 was reported to express high in SCC and low in AC opposite to miR-375. We evaluated miR-375 and CLDN1 expression levels by quantitative polymerase chain reaction (qPCR) and Western blotting in 12 NSCLC cell lines. The effect of miR-375 overexpression upon the CLDN1 expression was evaluated in 5 NSCLC cell lines by transfecting miR-375 precursor. It showed that the expression of CLDN1 messenger RNA and protein were attenuated by miR-375 overexpression. Luciferase reporter assay was performed to confirm direct interaction between miR-375 and CLDN1. We cloned 3′-UTR of CLDN1 cDNA into the downstream of a luciferase reporter gene and co-transfected this vector into A549 cells with miR-375 precursor. MiR-375 overexpression resulted in a 3-fold repression of luciferase activity (p &lt; 0.001). To ascertain the clinical validity, we analyzed the relationship between miR-375 and CLDN1 expression in 63 clinical samples of NSCLC. There was a negative correlation between miR-375 and CLDN1 expression (r = -0.35, p = 0.005). In addition, we analyzed the correlation between miR-375 expression and overall survival in the same samples. High miR-375 expression correlated with poor survival in NSCLC (p = 0.043). To investigate the reason why high miR-375 expression lead to poor survival, wound healing assay was performed to evaluate the effect of miR-375 overexpression on the cell migration in SK-MES-1 cells. The cell migration was promoted by miR-375 overexpression, suggesting the high potential of invasion and metastasis in NSCLC expressing high level of miR375. In conclusion, we found that CLDN1 is a novel target of miR-375, and high miR-375 expression leads to poor survival in NSCLC.

Citation Format: Satoshi Yoda, Kenzo Soejima, Hiroyuki Yasuda, Takashi Sato, Daisuke Arai, Keiko Ohgino, Kota Ishioka, Tetsuo Tani, Ayano Oashi, Aoi Kuroda, Makoto Nishino, Masayoshi Miyawaki, Junko Hamamoto, Katsuhiko Naoki, Tomoko Betsuyaku. Claudin-1, a novel target of miR-375 in non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5195. doi:10.1158/1538-7445.AM2014-5195