Circular RNA rno_circ_0004002 regulates cell proliferation, apoptosis, and epithelial-mesenchymal transition through targeting miR-342-5p and Wnt3a in anorectal malformations

Circular RNAs (circRNAs) are a novel class of noncoding RNAs that have regulatory functions in varieties of diseases. The purpose of this study was to investigate the potential role of circRNAs in anorectal malformations (ARM). With sequencing analysis, 78 circRNAs were identified to be differentially expressed on gestational day (GD) 19 in ARM and normal anorectal tissues, including 41 upregulated and 37 downregulated circRNAs. Among the downregulated circRNAs, rno_circ_0004002 was selected as the candidate circRNA. Functionally, silencing of rno_circ_0004002 was correlated with suppression of proliferation, and promotion of apoptosis and epithelial-mesenchymal transition (EMT) in anorectal development. For the mechanism investigation, bioinformatic prediction and luciferase reporter assay revealed that rno_circ_0004002 was proved to be a sponge of miR-342-5p, and miR-342-5p could target 3'-UTR of Wnt3a to negatively regulate its expression. The quantitative reverse transcription polymerase chain reaction (qRT-PCR) results showed that the expression of miR-342-5p was significantly increased in ARM anorectal tissues compared with normal tissues on GD19 and GD21, and the expression of Wnt3a demonstrated the opposite on GD21. In addition, rescue assays disclosed that miR-342-5p inhibitor could reverse the role of rno_circ_0004002 knockdown on the repression of anorectal development. In summary, our study shed light on the regulatory mechanism of rno_circ_0004002 in cell proliferation, apoptosis, and EMT via sponging miR-342-5p, which downregulated the Wnt3a expression in anorectal development. We also suggested that GD19 and GD21 was the crucial time in the progression of ARM, and rno_circ_0004002/miR-342-5p/Wnt3a might serve as potential therapeutic targets for ARM.