miRNA as Potential Biomarkers of Breast Cancer in the Lebanese Population and in Young Women: A Pilot Study Farah J. Nassar 1" , Maya El Sabban 2" , Nathalie K. Zgheib 3 , Arafat Tfayli 4 , Fouad Boulos 5 , Mark Jabbour 5 , Nagi S. El Saghir 4 , Rabih Talhouk 1 , Ali Bazarbachi 4 , George A. Calin 6 , Rihab Nasr 2 * 1 Department of Biology, Faculty of Arts and Sciences, American University of Beirut, Beirut, Lebanon, 2 Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut, Lebanon, 3 Department of Pharmacology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon, 4 Department of Internal Medicine, Faculty of Medicine, American University of Beirut, Beirut, Lebanon, 5 Department of Pathology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon, 6 Department of Experimental Therapeutics, Division of Cancer Medicine, University of Texas MD Anderson Cancer Center, Houston, Texas, United States of America Abstract Relative to western populations, the percentage of women diagnosed with breast cancer at a young age in Lebanon is high. While the younger age of the Lebanese population compared to the West certainly contributes to this difference, potential genetic, reproductive and/or biological factors likely play an important role. The objective of this study is to investigate the contribution of miRNAs in this setting through the analysis of the expression of five reported dysregulated miRNAs, miR- 148b, miR-10b, miR-21, miR-221, and miR-155 in 20 normal and 57 cancerous breast tissues from Lebanese breast cancer patients. After finding their relative expression by quantitative reverse transcription real time PCR, the results were analyzed with respect to the patients’ clinical and histopathology presentations. Compared to normal breast tissues, significant upregulation of miR-155, miR-21 and miR-148b, notable downregulation of miR-10b and non-significant expression of miR- 221 were observed in tumor tissues. Moreover, miR-10b was significantly underexpressed in estrogen/progesterone receptor (ER/PR) negative tumors relative to ER/PR positive tumor tissues. miR-155 was also significantly overexpressed in postmenopausal patients and in those of age at diagnosis greater than 40 years old as well as in PR negative or in human epidermal growth factor 2 (Her2) positive tissues. This study is the first one to report miRNA expression patterns in Lebanese breast cancer patients. We found that differential miRNA expression in breast cancer could be variable between Lebanese and Western populations. miR-10b was positively correlated with the ER and PR status and miR-155 could be a noteworthy biomarker for the menopausal state, age at diagnosis, PR and Her2 status. Hence, miRNA can be used as biomarkers for early breast cancer detection. Citation: Nassar FJ, El Sabban M, Zgheib NK, Tfayli A, Boulos F, et al. (2014) miRNA as Potential Biomarkers of Breast Cancer in the Lebanese Population and in Young Women: A Pilot Study. PLoS ONE 9(9): e107566. doi:10.1371/journal.pone.0107566 Editor: Soheil S. Dadras, University of Connecticut Health Center, United States of America Received January 17, 2014; Accepted August 19, 2014; Published September 18, 2014 Copyright: ß 2014 Nassar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Medical Practice Plan at the American University of Beirut. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Dr. George Calin is a co-author and a PLOS ONE Editorial Board member. However, this does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria. * Email: rn03@aub.edu.lb " These authors are co-first authors on this work. Introduction Breast cancer is one of the leading health concerns worldwide, affecting over one million women every year. In Lebanon, it is one of the most common type of cancer constituting about one third of all female cancers [1]. Interestingly, a significant number of Lebanese breast cancer patients were noted to be of young age at the time of diagnosis as 22% of the cases were below the age of 40 years old compared to 6% in the Western populations [2]. Moreover, Lebanese women who are diagnosed at young age (less than 35 years old) and in their premenopausal state were shown to present with a more aggressive disease and poorer survival in spite of adequate therapy [3]. The presence of signs of more aggressive features in breast cancer in young women, and the occurrence of breast cancer in young Lebanese women 10 years earlier in age than those in the West, strengthens the importance of determining the biological factors behind those differences, and perhaps revealing novel biomarkers for early screening and detection of breast cancer. Since the discovery of microRNA (miRNA) in C. elegans twenty years ago, this major subclass of non-coding RNA molecules act as gene modulators mostly at the posttranscriptional level by causing translation repression or degradation of mRNA [4]. miRNAs play diverse roles in normal cellular processes such as cell cycle, proliferation and apoptosis as well as in disease conditions including cancer, diabetes, neuro-degenerative disorder and cardiac hypertrophy [5,6]. miRNA was first correlated with breast cancer by Iorio and his colleagues. Using microarray analysis, they discovered a differential miRNA profile pattern between cancer- ous and normal breast tissues [7]. miRNAs were later shown to modulate tumor suppressor and oncogenic pathways thereby PLOS ONE | www.plosone.org 1 September 2014 | Volume 9 | Issue 9 | e107566