br Conclusions We identified the circRNA expression profile in

Conclusions: We identified the circRNA Cyclosporin H profile in ovarian cancer tissues and further verified the existence and expression of six randomly selected differentially expressed circRNAs. Besides, we also found that circHIPK3 is an important regulator of ovarian cancer progression.
1. Introduction
Ovarian cancer is the most fatal malignancy in gynecologic cancer, and 60% of total patients present at advanced stages (Siegel et al., 2018). Current detection methods include contrast-enhanced computed tomography imaging and the ovarian cancer antigen CA125 measure-ment, but these methods are incapable of examining the cancer at early
stages (Vaughan et al., 2011). The overall 5-year survival rate for pa-tients at all stages remains at only 47% but is as low as 29% at stages III-IV (Siegel et al., 2018). Optimal cytoreductive surgery combined with platinum-based chemotherapy is the first-line treatment. However, ovarian cancer relapse and subsequent resistance to chemotherapy lead to a high mortality rate (Vaughan et al., 2011). Therefore, effective therapies are urgently needed.
Abbreviations: NGS, next-generation sequencing; circRNAs, circular RNAs; EOC, epithelial ovarian cancer; NOT, normal ovarian tissues; qRT-PCR, quantitative real-time-PCR; RT-PCR, reverse-transcription PCR; CCK8, cell counting-kit 8; ceRNA, competing endogenous RNA; CDR1as, cerebellar degeneration-related 1 antisense transcript
Corresponding authors at: Department of Gynecology, Women’s Hospital of Nanjing Medical University (Nanjing Maternity and Child Health Care Hospital), No. 123, Tianfei Xiang, Mochou Road, Nanjing 210004, China. E-mail addresses: [email protected] (R. Shen), [email protected] (X. Jia).
1 These authors contributed equally to this work.
It has been reported that competing endogenous RNA (ceRNA) form a complicated regulatory network in cancer development and pro-gression (Salmena et al., 2011). As a novel class of ceRNA, circular RNAs (circRNA) are widely expressed in the mammalian transcriptome (Han et al., 2018). Currently, thousands of novel circRNAs have been identified by high-throughput sequencing and bioinformatic ap-proaches (Jeck and Sharpless, 2014). CircRNA commonly originate from head-to-tail back-splicing of pre-mRNA in which the 5’ and 3’ ends are covalently linked and loop structures are formed (Barrett and Salzman, 2006). Recent studies have revealed that circulation is fa-cilitated by reversed complementary sequences in the flanking introns as well as lariat introns and are modulated by RNA-binding proteins (Chen, 2016). Due to the lack of a 3’ polyadenylated tail, most circRNA can resist the digestion of the exonuclease, whereas their linear coun-terpart can be degraded (Zheng et al., 2016). CircRNAs are abundant and conserved among species, but expression is specifically regulated in different tissues and developmental stages (Barrett and Salzman, 2006). Exonic circRNAs predominantly distribute in the cytoplasm and mainly act as miRNA sponges (Chen, 2016). The most well-known exonic cir-cRNA, known as cerebellar degeneration-related 1 antisense transcript (CDR1as), harbors more than 70 binding sites of miR-7 and exerts its function primarily through sponging of miR-7 (Memczak et al., 2013).
In the current study, we investigated the expression profiles of circRNAs in epithelial ovarian cancer (EOC) and normal ovarian tissues (NOT) using RNA high-throughput sequencing. Six randomly selected circRNAs were examined in 18 EOC and 18 NOT and their expression was verified. Furthermore, we detected all six circRNAs in samples treated with RNase R and DNase, which could digest linear RNAs and genomic DNA (gDNA), respectively. The head-to-tail splicing was ver-ified by Sanger sequencing. CircHIPK3 (hsa_circ_0000284/ hsa_circRNA001123), which represents the most abundant circRNA in our data, was remarkably downregulated in EOC. Our study also found that circHIPK3 expression was significantly associated with prolifera-tion, migration and invasion of ovarian cancer cells and normal ovarian epithelial cells. Therefore, circRNAs might play important roles in ovarian cancer progression.