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.