Research Article Circulating PIWI-Interacting RNAs piR-5937 and piR-28876 Are Promising Diagnostic Biomarkers of Colon Cancer Petra Vychytilova-Faltejskova 1,2 , Karolina Stitkovcova 1 , Lenka Radova 1 , Milana Sachlova 2 , Zdenka Kosarova 1 , Katerina Slaba 1 , Zdenek Kala 1,3 , Marek Svoboda 1,2 , Igor Kiss 2 , Rostislav Vyzula 2 , William C. Cho 4 , and Ondrej Slaby 1,2 Abstract Background: The early detection of colon cancer is one of the main prerequisites for successful treatment and mortality reduction. Circulating PIWI-interacting RNAs (piRNA) were recently identified as novel promising biomarkers. The pur- pose of the study was to assess the profiles of piRNAs in blood serum of colon cancer patients with the aim to identify those with high diagnostic potential. Methods: Blood serum samples from 403 colon cancer patients and 276 healthy donors were included in this 3-phase biomarker study. Large-scale piRNA expression profiling was performed using Illumina small RNA sequencing. The diagnos- tic potential of selected piRNAs was further validated on inde- pendent training and validation sets of samples using RT-qPCR. Results: In total, 31 piRNAs were found to be significantly deregulated in serum of cancer patients compared with healthy donors. Based on the levels of piR-5937 and piR- 28876, it was possible to differentiate between cancer patients and healthy donors with high sensitivity and specificity. More- over, both piRNAs exhibited satisfactory diagnostic perfor- mance also in patients with stage I disease and enabled detection of colon cancer with higher sensitivity than currently used biomarkers CEA and CA19-9. Finally, the expression of analyzed piRNAs in blood restored significantly 1 month after the surgical resection. Conclusions: Based on our findings, piRNAs are abundant in human blood serum. Furthermore, their levels in colon cancer have been observed to be significantly deregulated. However, their involvement in carcinogenesis must be further established. Impact: piRNAs could serve as promising noninvasive bio- markers for early detection of colon cancer. Cancer Epidemiol Biomarkers Prev; 27(9); 1019–28. Ó2018 AACR. Introduction Colon cancer accounts for approximately for 7% of all cancers, and it is one of the most common causes of cancer-related deaths. The prognosis of patients depends mostly on tumor–node– metastasis (TNM) stage at the time of diagnosis as well as on the possibility of curative surgical resection, which can be accom- plished only in patients with localized disease (1). Thus, the early detection of colon cancer or, even better, precancerous lesions is one of the main prerequisites for mortality reduction and more successful treatment. In addition, novel biomarkers suitable for monitoring of disease progression as well as treatment response are in great demand. Currently, fecal occult blood testing and endoscopic app- roaches are the predominant screening methods used for early detection of colon cancer. Although it has been shown that these methods significantly contribute to reduced risk of colon cancer– associated mortality (2), screening effectiveness is limited by test performance, high costs, and invasiveness as well as a suboptimal screening compliance. Thus, the development of a simple blood- based test, with a specimen drawn during the routine medical check-up, could improve the screening rates. Lately, various molecules including DNA (3), proteins (4), mRNA (5), or miR- NAs (6, 7) have shown a great potential to serve as new molecular markers for the development of noninvasive and accurate tests for colon cancer screening. Recently, several studies have indicated the abundance of novel class of small noncoding RNAs called PIWI-interacting RNAs (piRNA) in various types of body fluids (8, 9). These 26-32 nucleotide-long molecules have been shown to participate in the epigenetic regulation of cancer and other diseases and are key elements of cellular homeostasis (10). Furthermore, they play an important role in tumor formation, proliferation, and migration of the cells (11, 12). Similar to miRNAs, piRNAs posttranscrip- tional regulation occurs in the cytoplasm. The piRISC protects the integrity of the genome by binding the transposable elements as well as mRNAs or lncRNAs (13). Moreover, these molecules may regulate gene expression through histone modifications and DNA methylation (14). Unlike miRNAs, piRNAs are extremely diverse, with at least hundreds of thousands of mature species transcribed from thousands of genomic loci (15). However, similarly to 1 Centre for Molecular Medicine, Central European Institute of Technology, Masaryk University, Brno, Czech Republic. 2 Department of Comprehensive Cancer Care, Masaryk Memorial Cancer Institute, Faculty of Medicine, Masaryk University, Brno, Czech Republic. 3 Department of Surgery, Institutions Shared with the Faculty Hospital Brno, Faculty of Medicine, Masaryk University, Brno, Czech Republic. 4 Cancer Research Unit, Department of Clinical Oncology, Queen Elizabeth Hospital, Kowloon, Hong Kong, PR China. Note: Supplementary data for this article are available at Cancer Epidemiology, Biomarkers & Prevention Online (http://cebp.aacrjournals.org/). Corresponding Author: Ondrej Slaby, Masaryk University, Kamenice 753/5, Brno 625 00, Czech Republic. Phone: 00420549496876; E-mail: ondrej.slaby@ceitec.muni.cz doi: 10.1158/1055-9965.EPI-18-0318 Ó2018 American Association for Cancer Research. Cancer Epidemiology, Biomarkers & Prevention www.aacrjournals.org 1019 on May 28, 2020. © 2018 American Association for Cancer Research. cebp.aacrjournals.org Downloaded from Published OnlineFirst July 5, 2018; DOI: 10.1158/1055-9965.EPI-18-0318