Basic Study
Copyright ©The Author(s) 2020. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 7, 2020; 26(29): 4272-4287
Published online Aug 7, 2020. doi: 10.3748/wjg.v26.i29.4272
Rno_circ_0005139 regulates apoptosis by targeting Wnt5a in rat anorectal malformations
Dan Liu, Yuan Qu, Zheng-Nong Cao, Hui-Min Jia
Dan Liu, Yuan Qu, Zheng-Nong Cao, Hui-Min Jia, Department of Pediatric Surgery, Shengjing Hospital of China Medical University, Shenyang 110004, Liaoning Province, China
Author contributions: Liu D and Qu Y performed the experiments, and analyzed the results; Liu D drafted the manuscript; Cao ZN and Jia HM reviewed manuscript; Cao ZN supervised the manuscript; Jia HM revised the final manuscript; all authors revised and approved the final manuscript.
Supported by the National Natural Science Foundation of China, No. 81671503.
Institutional review board statement: The study was reviewed and approved by Medical Research and New Technology Ethics Committee of Shengjing Hospital, affiliated with China Medical University.
Institutional animal care and use committee statement: All animal experiments conformed to the internationally accepted principles for the care and use of laboratory animals (licence No. 2016PS045K).
Conflict-of-interest statement: All other authors have nothing to disclose.
ARRIVE guidelines statement: The authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Hui-Min Jia, PhD, Chief Doctor, Professor, Department of Pediatric Surgery, Shengjing Hospital of China Medical University, No. 36 Sanhao Street, Heping District, Shenyang 110004, Liaoning Province, China.
Received: February 7, 2020
Peer-review started: February 7, 2020
First decision: April 22, 2020
Revised: May 9, 2020
Accepted: May 26, 2020
Article in press: May 26, 2020
Published online: August 7, 2020

The molecular mechanisms underlying anorectal malformations (ARM) are not fully established. Circular RNAs (circRNAs) are new born non-coding RNAs, and their role in ARM is unclear. We assumed that rno_circ_0005139 influences apoptosis and proliferation by acting as a miR-324-3p sponge, and downregulating Wnt5a in ARM.


To identify the differential expression of circRNAs and mRNAs in a rat ARM model.


Sixty-six pregnant Wistar rats were randomly divided into two groups: ARM group (2-imidazolidinethione-induced) and control groups. Embryos were harvested by cesarean delivery, and anorectal tissue was taken on embryonic days 16 (E16), 17 (E17), 19 (E19), and 21 (E21). RNA sequencing and gene microarray analysis was used to identify differentially expressed circRNAs and mRNAs in the ARM in a rat model. We selected 6 circRNAs and 3 mRNAs in the Wnt signal pathway from the result of the RNA sequencing and gene microarray analysis, and quantitative reverse transcription polymerase chain reaction was performed to evaluate their tissue expression. According to bioinformatics prediction, rno_circ_0005139 acted as a miR-324-3p sponge to regulate the expression of Wnt5a. We chose rno_circ_0005139 and Wnt5a as the final candidates. We tested the function of rno_circ_0005139 and the binding sites between rno_circ_0005139 and miR-324-3p, miR-324-3p and Wnt5a by luciferase assays. Co-transfection of rno_circ_0005139 and miR-324-3p was to verify their functional consistency.


We identified 38 upregulated and 42 downregulated circRNAs on E17 (P < 0.05), and 301 mRNAs were upregulated and 256 downregulated in the ARM on E17 (P < 0.05, fold-change > 2.0). We found that rno_circ_0006880 and rno_circ_0011386 were upregulated, whereas rno_circ_0000436, rno_circ_0005139, rno_circ_0009285, rno_circ_0014367, Wnt5a, Wnt10b, and Wnt2b were downregulated in ARM tissues. According to bioinformatics prediction, rno_circ_0005139 acted as a miR-324-3p sponge to regulate the expression of Wnt5a. We chose rno_circ_0005139 and Wnt5a as the final candidates. Because the role and molecular mechanism of rno_circ_0005139 are poorly understood, its effect on apoptosis and proliferation was investigated by in vitro plasmid transfection. A luciferase experiment showed that rno_circ_0005139 could bind with miR-324-3p, which negatively regulated Wnt5a expression. The expression of miR-324-3p was significantly higher in ARM anorectal tissues than that in control group on E17 and E19; Wnt5a expression showed the opposite trend. In addition, a miR-324-3p inhibitor attenuated the effects of rno_circ_0005139 knockdown on ARM development.


Rno_circ_0005139 influences cell proliferation and apoptosis by acting as a miR-324-3p sponge, thereby downregulating Wnt5a in ARM. Accordingly, rno_circ_0005139, miR-324-3p, and Wnt5a could be targeted therapeutic factors for ARM.

Keywords: Anorectal malformation, Circular RNA, MicroRNA, Wnt5a, Rno_circ_0005139

Core tip: Rno_circ_0005139, miR-324-3p, and Wnt5a play regulatory roles in the pathogenesis of anorectal malformations (ARM). Rno_circ_0005139 is a potential biomarker or therapeutic target in the ARM. In general, this study provides an important basis for further studies on the diagnosis, treatment, and prevention of ARM.