Multiplex bioimaging of piRNA molecular pathway-regulated theragnostic effects in a single breast cancer cell using a piRNA molecular beacon Youn Jung Lee a, 1 , Sung Ung Moon a, 1 , Min Geun Park c, 1 , Woon Yong Jung d, 1 , Yong Keun Park c , Sung Kyu Song c , Je Gyu Ryu c , Yong Seung Lee a , Hye Jung Heo a , Ha Na Gu a , Su Jeong Cho a , Bahy A. Ali e, f , Abdulaziz A. Al-Khedhairy e , Ilkyun Lee c, ** , Soonhag Kim a, b, * a Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Gangneung-si, Gangwon-do, 270-701, Republic of Korea b Catholic Kwandong University International St. Mary's Hospital, Incheon Metropolitan City, 404-834, Republic of Korea c Department of Surgery, Catholic Kwandong University International St. Mary's Hospital, Incheon Metropolitan City, 404-834, Republic of Korea d Department of Pathology, Catholic Kwandong University International St. Mary's Hospital, Incheon Metropolitan City, 404-834, Republic of Korea e Al-Jeraisy DNA Research Chair, Department of Zoology, College of Science, King Saud University, Riyadh,11451, Saudi Arabia f Department of Nucleic Acids Research, Genetic Engineering and Biotechnology Research Institute, City for Scientific Research and Technological Applications, Alexandria, Egypt article info Article history: Received 16 February 2016 Received in revised form 18 May 2016 Accepted 24 May 2016 Available online 1 June 2016 Keywords: Molecular beacon piRNA-36026 Cancer theragnosis Multiplex bioimaging Molecular pathway abstract Recently, PIWI-interacting small non-coding RNAs (piRNAs) have emerged as novel cancer biomarkers candidate because of their high expression level in various cancer types and role in the control of tumor suppressor genes. In this study, a novel breast cancer theragnostics probe based on a single system targeting the piRNA-36026 (piR-36026) molecular pathway was developed using a piR-36026 molecular beacon (MB). The piR-36026 MB successfully visualized endogenous piR-36026 biogenesis, which is highly expressed in MCF7 cells (a human breast cancer cell line), and simultaneously inhibited piR- 36026-mediated cancer progression in vitro and in vivo. We discovered two tumor suppressor proteins, SERPINA1 and LRAT, that were directly regulated as endogenous piR-36026 target genes in MCF7 cells. Furthermore, multiplex bioimaging of a single MCF7 cell following treatment with piR-36026 MB clearly visualized the direct molecular interaction of piRNA-36026 with SERPINA1 or LRAT and subsequent molecular therapeutic responses including caspase-3 and PI in the nucleus. © 2016 Elsevier Ltd. All rights reserved. 1. Introduction Bioimaging is revolutionizing our ability to see and monitor specific proteins and genes, and ultimately characterize molecular pathways within living organisms [1]. This method offers many technical advantages over traditional techniques, as it is rapid, non- invasive and less labor-intensive than pathology and chemistry- based assays while allowing imaging at the cellular and molecu- lar level in living animals, in real-time and with a truly quantitative outcome [2]. However, single biomarker imaging of diseases cannot represent the various mechanisms and phases of neoplastic path- ogenesis caused by complex gene interactions. In contrast, imaging along a molecular regulatory pathway offers diagnosis sensitivity and accuracy for various diseases by targeting different epitopes expressed in a single cell or a wide range of diseases. Typically, PIWI-interacting RNAs (piRNAs) are small, 21e30 nt single-stranded noncoding RNAs that associate with PIWI proteins in the male germline [3]. piRNAs are involved in maintaining genome stability by suppressing transposon activity, assembly of the telomere protection complex, RNA silencing and epigenetic control of gene expression by establishment of a repressive * Corresponding author. Institute for Bio-Medical Convergence, College of Med- icine, Catholic Kwandong University, Gangneung-si, Gangwon-do, 270-701, Re- public of Korea ** Corresponding author. Department of Surgery, College of Medicine, Catholic Kwandong University, Gangneung-si, Gangwon-do, 270-701, Republic of Korea. Tel.: þ82 32 290 2771; fax: þ82 32 280 2774. E-mail addresses: iklee68@gmail.com (I. Lee), kimsoonhag@empas.com (S. Kim). 1 These authors contributed equally to this work. Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials http://dx.doi.org/10.1016/j.biomaterials.2016.05.052 0142-9612/© 2016 Elsevier Ltd. All rights reserved. Biomaterials 101 (2016) 143e155