OncoTargets and Therapy Dovepress open access to scientific and medical research Open Access Full Text Article ORIGINAL RESEARCH A Combination of circRNAs as a Diagnostic Tool for Discrimination of Papillary Thyroid Cancer This article was published in the following Dove Press journal: OncoTargets and Therapy Erlan Shi Background and Aim: Circular RNAs (circRNAs) have been highlighted to exert essential Jun Ye biological functions in papillary thyroid cancer (PTC). The purpose of this study was explore Rong Zhang diagnostic utility of circRNAs in PTC patients. fi Shuai Ye Patients and Methods: The distinctive expression pro le of serum circRNAs was deter- Suwan Zhang mined by individual quantitative real-time PCR (qRT-PCR) in two independent cohorts of 113 PTC patients, 80 thyroid nodules, and 111 healthy controls (HCs). A combination of Yunsheng Wang circRNAs (circRNA-based combination index) was constructed by logistic regression. Yonghong Cao Results: Individual qRT-PCR identification showed that two circRNAs (circRAPGEF5 and Wu Dai hsa_circ_0058124) were significantly up-regulated in PTC patients compared with HCs and Department of Endocrinology, Hefei thyroid nodules. Receiver-operating characteristic (ROC) curve analysis suggested that fi Hospital Af liated to Anhui Medical a combination of circRNAs was superior to individual circRNA in distinguishing PTC University, The Second People’s Hospital of Hefei, Hefei 230011, People’s Republic patients from HCs and thyroid nodules with area under ROC curve of more than 0.80. of China Furthermore, the combination of circRNAs increased significantly after systematic treatment, suggesting that it could monitor PTC dynamics. Additionally, the combination of circRNAs was independently correlated with PTC presence. Conclusion: The combination of these altered circRNAs was correlated with PTC and may serve as a novel diagnostic approach. Keywords: circular RNA (circRNA), papillary thyroid cancer (PTC), thyroid nodule, diagnosis, circRAPGEF5, hsa_circ_0058124 Introduction Thyroid cancer (TC) is one of the common endocrine malignancies worldwide, and its incidence has increased rapidly over recent decades, especially papillary TC (PTC).1,2 PTC is the most common histological type of TC and accounts for about 85% of all TCs. Despite synchronous lymph node metastases in around 30% of PTCs, its 5-year overall survival (OS) rate is almost 98% (Howlader et al, SEER Cancer Statistics Review, 1975–2012. National Cancer Institute, Bethesda, 2015). However, only 59% of patients with progressed TC have a 5-year OS rate.3 Although fine-needle aspiration (FNA) presently guides therapeutic strategies of patients with a thyroid nodule, the prevalence of indeterminate and non-diagnostic cytology from FNA is still high at around 30%.3 Therefore, more efforts are Correspondence: Wu Dai; Yonghong Cao Department of Endocrinology, Hefei focused on trying to identify a new approach for the clinical assessment of Hospital Affiliated to Anhui Medical advanced cancer. University, The Intersection of Guangde Road and Leshui Road, Hefei 230011, Amounting evidence shows that epigenetic molecular markers may improve clin- People’s Republic of China ical assessment of many diseases.4,5 Various circular RNAs (circRNAs) have been Email [email protected]; 6 fi[email protected] verified by genome-wide analyses, which are involved in many pathological processes submit your manuscript | www.dovepress.com OncoTargets and Therapy 2020:13 4365–4372 4365 DovePress © 2020 Shi et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php – http://doi.org/10.2147/OTT.S247796 and incorporate the Creative Commons Attribution Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Shi et al Dovepress through transcriptional and post-transcriptional regulatory A total of 113 PTC patients, 80 patients with thyroid mechanisms.7 However, the system characteristics of nodules and 111 HCs were collected in this study and circRNAs in PTC patients remain unclear. Previous reports randomly divided into the training and test cohorts. The have demonstrated that circRAPGEF5, hsa_circ_0058124, training cohort included 52 PTC patients, 38 patients with circRNA_102171 and circBACH2 contribute to PTC prolif- thyroid nodules and 50 HCs to screen nominated circRNAs. eration and metastasis though a similar mechanism of com- The test cohort enrolled 61 PTC patients, 42 thyroid 8–10 fi peting endogenous RNA. However, clinical significance nodules and 61 HCs to con rm the expression of screened of these circRNAs remains unknown. circRNAs and their potential association with the risk of In this study, we firstly detected the differential expression PTC. In brief, a total of 113 serum samples were collected of four circRNAs in the training cohort of 52 PTC patients, from all participants with PTC receiving surgery at Hefei fi 38 patients with thyroid nodules and 50 healthy controls Hospital af liated to Anhui Medical University and The ’ fi (HCs). We then confirmed these findings in the test cohort of People s Hospital of Chizhou with a de nite pathological 61 PTC patients, 42 patients with thyroid nodules and 61 HCs, diagnosis of PTC during surgery. Patients with PTC did not undergo radiotherapy, chemotherapy, and blood transfusion and showed their temporal profiles at pre- and post-treatment. before surgery. Clinical features of all enrolled patients were Furthermore, we investigated the combined diagnostic and recorded including sex, age, and tumor staging in Table 1. predictive power of circRAPGEF5 and hsa_circ_0058124 in PTC patients. Blood Samples Patients and Methods Blood samples were obtained from 113 PTC patients at 2 h before surgery and 7 days after operation, respectively. Patients and Samples Furthermore, we collected 63 samples from PTC patients after The study design was approved by the Medical Ethics treatment. A total of 113 samples were collected from newly fi Committee of Hefei Hospital af liated to Anhui Medical diagnosed PTC patients, and 80 patients with thyroid University (Hefei, China). We obtained the written informed nodules were also collected. Additionally, 111 healthy volun- consent from all enrolled participants. Tissue and blood teers were enrolled as the referred group who were from samples were processed at Hefei Hospital affiliated to medical examination without evidence of malignancies. Anhui Medical University and followed with ethical and Serum samples were isolated from blood samples after being legal standards. This study was also conducted in accordance centrifuged at 3000 rpm for 10 min and were stored at −80 ° with the Declaration of Helsinki. Cuntil RNA extraction. Table 1 Clinical Characteristics of Patients Enrolled in the Training and Test Cohorts Clinical Training Cohort Test Cohort Characteristics PTC Thyroid Nodule HC P PTC Thyroid Nodule HC P (n=52) (n=38) (n=50) (n=61) (n=42) (n=61) Age (years)* 64.2±4.56 65.0±5.67 64.9 0.277 65.5±6.68 66.4±7.83 64.1 0.432 ±5.32 ±6.77 Gender (male/female) 27/25 20/18 20/30 0.381 29/32 22/20 25/36 0.508 TNM staging I–II 35 (67.31) –––38 (62.30) ––– III–IV 17 (32.69) –––23 (37.70) ––– Multifocality 18 (34.62) –––20 (32.79) ––– Lymph node metastasis 32 (61.54) –––35 (57.38) ––– Distant metastasis 4 (7.69) –––5 (8.20) ––– Note: *Data were expressed as mean ± S.D. Abbreviations: PTC, papillary thyroid cancer; HC, healthy control. 4366 submit your manuscript | www.dovepress.com OncoTargets and Therapy 2020:13 DovePress Dovepress Shi et al RNA Preparation and Quantitative of circRNAs were determined by Youden’s index. Logistic Real-Time PCR regression model was used to determine circle RNAs’ regression coefficient (0.392 for circRAPGEF5 and 0.792 Total RNA was extracted from serum samples using TRIzol for hsa_circ_0058124), and a combination of circRNAs reagent (Invitrogen, MA, USA) as described previously.4 was calculated on this equation (0.392×ΔCt RNA integrity was determined by agarose gel electrophor- circRAPGEF5 +0.792×ΔCt ). The predictive ability of esis (1% gel). cDNA was synthesized using the RNA-to- hsa_circ_0058124 circlRNAs was further analyzed by logistic regression cDNA kit (Takara Vio, Dalian, China) according to the analyses. P<0.05 was considered statistically significant. manufacturer’s instruction. CircRNA relative expression was determined using the ABI 7500 System (Applied Biosystems, USA) and a SYBR Green kit (Takara, Dalian, Results China) with the following qPCR cycling program: 45 cycles Clinical Characteristics of the Study including denaturation at 95°C for 5 sec, annealing at 60 for Participants 30 sec and extension at 72°C for 30 sec. GAPDH was A total of 52 patients with PTC, 38 patients with thyroid considered as an internal control and RNA expression was nodules and 50 HCs were enrolled in the training cohort to Δ calculated using the Ct method. All primers were synthe- screen the nominated circRNAs. To further verify the sized by Sangon Biotech Co. Ltd. (Shanghai, China). expression of screened circRNAs and their potential asso- ciation with the risk of PTC, we conducted a case-control Statistical Analysis