Abstract
Anorectal malformations (ARMs) are among the most common congenital terminal digestive tract malformations. Circular RNAs (circRNAs), a novel type of endogenous non-coding RNAs, play roles in the development of the digestive system; however, their contributions to the pathogenesis of ARMs are not well-established. In this study, we explored the mechanism underlying ethylenethiourea (ETU)-induced ARMs by profiling circRNA expression via RNA-seq and constructing a regulatory circRNA-miRNA-mRNA network. Nine pregnant rats were gavage-fed a single dose of 125 mg/kg 1% ETU (ARM group) on gestational day 10 (GD10), and another 9 pregnant rats received a similar dose of saline (normal group) as a control. Embryos were obtained by cesarean section on the key time-points of anorectal development (GD14, GD15, and GD16). Hindgut samples isolated from the fetuses were evaluated by high-throughput sequencing and differentially expressed circRNAs were validated by reverse transcription-quantitative polymerase chain reaction, agarose gel electrophoresis, and Sanger cloning and sequencing. A total of 18295 circRNAs were identified in the normal and ARM groups. Based on the 425 differentially expressed circRNAs (|Fc| > 2, p < 0.05), circRNA-miRNA and miRNA-mRNA pairs were predicted using miREAP, miRanda, and TargetScan. A total of 55 circRNAs (14 up- and 41 downregulated in the ARM group compared to the normal group) were predicted to bind to 195 miRNAs and 947 mRNAs. Competing endogenous RNA networks and a Kyoto Encyclopedia of Genes and Genomes analysis revealed that novel_circ_001042 had the greatest connectivity and was closely related to ARM-associated signaling pathways, such as the Wingless Type MMTV integration site family, mitogen-activated protein kinase, and transforming growth factor-β pathways. These results provide original insight into the roles of circRNAs in ARMs and provide a valuable resource for further analyses of molecular mechanisms and signaling networks.
Highlights
Anorectal malformations (ARMs) are the most common congenital terminal digestive tract malformations, with an incidence of approximately 1 in 5,000 births and a slightly lower incidence in females than in males (Cuschieri and Eurocat Working Group, 2001; Wood and Levitt, 2018)
9 pregnant rats were gavage-fed a single dose of 125 mg/kg 1% ETU (SigmaAldrich; Merck Millipore, Darmstadt, Germany) on gestational day 10 (GD10), and the remaining pregnant rats received the same dose of saline without ETU as a control
We found that the expression levels of 60 circRNAs were higher in the normal group and 42 were higher in the ARM group (Figure 4C), whereas levels of 33 circRNAs were lower in the normal group and 47 were lower in the ARM group (Figure 4D)
Summary
Anorectal malformations (ARMs) are the most common congenital terminal digestive tract malformations, with an incidence of approximately 1 in 5,000 births and a slightly lower incidence in females than in males (Cuschieri and Eurocat Working Group, 2001; Wood and Levitt, 2018). The wide spectrum of ARM phenotypes includes stenotic anus, ectopic anus, rectourethral fistula, and rectovestibular fistula (Endo et al, 1999; Bai et al, 2004; Wijers et al, 2014). More than half of patients with ARM suffer from congenital anomalies of the urogenital system, cardiovascular system, skeleton, and gastrointestinal tract (Stoll et al, 2007; Nah et al, 2012). It is essential to investigate the pathogenesis of ARMs
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
